Human DNA mismatch repair proteins

ABSTRACT

The present invention discloses three human DNA repair proteins and DNA (RNA) encoding such proteins and a prodeudre for producing such proteins by recombinant techniques. One of the human DNA repair proteins, hMLH1, has been mapped to chromosome 3 while hMLH2 has been mapped to chromosome 2 and hMLH3 has been mapped to chromosome 7. The polynucleotide sequences of the DNA repair proteins may be used for therapeutic and diagnostic treatments of a hereditary susceptibility to cancer.

[0001] This application is a divisional of, and claims the benefit ofpriority under 35 U.S.C. § 120 of, U.S. patent application Ser. No.08/468,024 filed Jun. 6, 1995, which is a continuation-in-part of, andclaims the benefit of priority under 35 U.S.C. § 120 of, InternationalApplication No. PCT/US95/01035 filed Jan. 25, 1995; U.S. patentapplication Ser. No. 08/468,024 filed Jun. 6, 1995 is also acontinuation-in-part of, and claims the benefit of priority under 35U.S.C. § 120 of, U.S. patent application Ser. No. 08/294,312 filed Aug.23, 1994, which is a continuation-in-part of, and claims the benefit ofpriority under 35 U.S.C. § 120 of, U.S. patent application Ser. No.08/210,143 filed Mar. 16, 1994, which is a continuation-in-part of, andclaims the benefit of priority under 35 U.S.C. § 120 of, U.S. patentapplication Ser. No. 08/187,757 filed Jan. 27, 1994; and thisapplication is also a divisional of, and claims the benefit of priorityunder 35 U.S.C. § 120 of, U.S. patent application Ser. No. 08/465,679filed Jun. 6, 1995, which is a continuation-in-part of, and claims thebenefit of priority under 35 U.S.C. § 120 of, U.S. patent applicationSer. No. 08/294,312 filed Aug. 23, 1994, which is a continuation-in-partof, and claims the benefit of priority under 35 U.S.C. § 120 of, U.S.patent application Ser. No. 08/210,143 filed Mar. 16, 1994, which is acontinuation-in-part of, and claims the benefit of priority under 35U.S.C. § 120 of, U.S. patent application Ser. No. 08/187,757 filed Jan.27, 1994; and this application is also a continuation-in-part of, andclaims the benefit of priority under 35 U.S.C. § 120 of, U.S. patentapplication Ser. No. 08/294,312 filed Aug. 23, 1994, which is acontinuation-in-part of, and claims the benefit of priority under 35U.S.C. § 120 of, U.S. patent application Ser. No. 08/210,143 filed Mar.16, 1994, which is a continuation-in-part of, and claims the benefit ofpriority under 35 U.S.C. § 120 of, U.S. patent application Ser. No.08/187,757 filed Jan. 27, 1994; and this application is also acontinuation-in-part of, and claims the benefit of priority under 35U.S.C. § 120 of, U.S. patent application Ser. No. 08/210,143 filed Mar.16, 1994, which is a continuation-in-part of, and claims the benefit ofpriority under 35 U.S.C. § 120 of, U.S. patent application Ser. No.08/187,757 filed Jan. 27, 1994; and this application is also acontinuation-in-part of, and claims the benefit of priority under 35U.S.C. § 120 of, U.S. patent application Ser. No. 08/187,757 filed Jan.27, 1994. Each of the aforementioned U.S. and International patentapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptides ofthe present invention are human homologs of the prokaryotic mutL4 geneand are hereinafter referred to as hMLH1, hMLH2 and hMLH3.

[0003] In both prokaryotes and eukaryotes, the DNA mismatch repair geneplays a prominent role in the correction of errors made during DNAreplication and genetic recombination. The E.coli methyl-directed DNAmismatch repair system is the best understood DNA mismatch repair systemto date. In E.coli, this repair pathway involves the products of themutator genes mutS, mutL, mutH, and uvrD. Mutants of any one of thesegenes will reveal a mutator phenotype. MutS is a DNA mismatch-bindingprotein which initiates this repair process, uvrD is a DNA helicase andMutH is a latent endonuclease that incises at the unmethylated strandsof a hemi-methylated GATC sequence. MutL protein is believed torecognize and bind to the mismatch-DNA-MutS-MutH complex to enhance theendonuclease activity of MutH protein. After the unmethylated DNA strandis cut by the MutH, single-stranded DNA-binding protein, DNA polymeraseIII, exonuclease I and DNA ligase are required to complete this repairprocess (Modrich P., Annu. Rev. Genetics, 25:229-53 (1991)).

[0004] Elements of the E.coli MutLHS system appears to be conservedduring evolution in prokaryotes and eukaryotes. Genetic study analysissuggests that Saccharomyces cerevisiae has a mismatch repair systemsimilar to the bacterial MutLHS system. In S. cerevisiae, at least twoMutL homologs, PMS1 and MLH1, have been reported. Mutation of either oneof them leads to a mitotic mutator phenotype (Prolla et al, Mol. Cell.Biol. 14:407-415 (1994)). At least three MutS homologs have been foundin S.cerevisiae, namely MSH1, MSH2, and MSH3. Disruption of the MSH2gene affects nuclear mutation rates. Mutants in S. cerevisae, MSH2,PMS1, and MLH1 have been found to exhibit increased rates of expansionand contraction of dinucleotide repeat sequences (Strand et al., Nature,365:274-276 (1993)).

[0005] It has been reported that a number of human tumors such as lungcancer, prostate cancer, ovarian cancer, breast cancer, colon cancer andstomach cancer show instability of repeated DNA sequences (Han et al.,Cancer, 53:5087-5089 (1993); Thibodeau et al., Science 260:816-819(1993); Risinger et al., Cancer 53:5100-5103 (1993)). This phenomenonsuggests that lack of the DNA mismatch repair is probably the cause ofthese tumors.

[0006] Little was known about the DNA mismatch repair system in humansuntil recently, the human homolog of the MutS gene was cloned and foundto be responsible for hereditary nonpolyposis colon cancer (HNPCC),(Fishel et al., Cell, 75:1027-1038 (1993) and Leach et al., Cell,75:1215-1225 (1993)). HNPCC was first linked to a locus at chromosome2p16 which causes dinucleotide instability. It was then demonstratedthat a DNA mismatch repair protein (MutS) homolog was located at thislocus, and that C→T transitional mutations at several conserved regionswere specifically observed in HNPCC patients. Hereditary nonpolyposiscolorectal cancer is one of the most common hereditable diseases of man,affecting as many as one in two hundred individuals in the westernworld.

[0007] It has been demonstrated that hereditary colon cancer can resultfrom mutations in several loci. Familial adenomatosis polyposis coli(APC), linked to a gene on chromosome 5, is responsible for a smallminority of hereditary colon cancer. Hereditary colon cancer is alsoassociated with Gardner's syndrome, Turcot's syndrome, Peutz-Jaegherssyndrome and juvenile polyposis coli. In addition, hereditarynonpolyposis colon cancer may be involved in 5% of all human coloncancer. All of the different types of familial colon cancer have beenshown to be transmitted by a dominant autosomal mode of inheritance.

[0008] In addition to localization of HNPCC, to the short arm ofchromosome 2, a second locus has been linked to a pre-disposition toHNPCC (Lindholm, et al., Nature Genetics, 5:279-282 (1993)). A stronglinkage was demonstrated between a polymorphic marker on the short armof chromosome 3 and the disease locus.

[0009] This finding suggests that mutations on various DNA mismatchrepair proteins probably play crucial roles in the development of humanhereditary diseases and cancers.

[0010] HNPCC is characterized clinically by an apparent autosomaldominantly inherited predisposition to cancer of the colon, endometriumand other organs. (Lynch, H. T. et al., Gastroenterology, 104:1535-1549(1993)). The identification of markers at 2p16 and 3p21-22 which werelinked to disease in selected HNPCC kindred unequivocally establishedits mendelian nature (Peltomaki, P. et al., Science, 260:810-812(1993)). Tumors from HNPCC patients are characterized by widespreadalterations of simple repeated sequences (microsatellites) (Aaltonen, L.A., et al., Science, 260:812-816 (1993)). This type of geneticinstability was originally observed in a subset (12 to 18% of sporadiccolorectal cancers (Id.). Studies in bacteria and yeast indicated that adefect in DNA mismatch repair genes can result in a similar instabilityof microsatellites (Levinson, G. and Gutman, G. A., Nuc. Acids Res.,15:5325-5338 (1987)), and it was hypothesized that deficiency inmismatched repair was responsible for HNPCC (Strand, M. et al., Nature,365:274-276 (1993)). Analysis of extracts from HNPCC tumor cell linesshowed mismatch repair was indeed deficient, adding definitive supportto this conjecture (Parsons, R. P., et al., Cell, 75:1227-1236 (1993)).As not all HNPCC kindred can be linked to the same loci, and as at leastthree genes can produce a similar phenotype in yeast, it seems likelythat other mismatch repair genes could play a role in some cases ofHNPCC.

SUMMARY OF THE INVENTION

[0011] hMLH1 is most homologous to the yeast mutL-homolog yMLH1 whilehMLH2 and hMLH3 have greater homology to the yeast mutL-homolog yPMS1(hMLH2 and hMLH3 due to their homology to yeast PMS1 gene are sometimesreferred to in the literature as hPMS1 and hPMS2). In addition to hMLH1,both the hMLH2 gene on chromosome 2q32 and the hMLH3 gene, on chromosome7p22, were found to be mutated in the germ line of HNPCC patients. Thisdoubles the number of genes implicated in HNPCC and may help explain therelatively high incidence of this disease.

[0012] In accordance with one aspect of the present invention, there areprovided novel putative mature polypeptides which are hMLH1, hMLH2 andhMLH3, as well as biologically active and diagnostically ortherapeutically useful fragments, analogs and derivatives thereof. Thepolypeptides of the present invention are of human origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding such polypeptides,including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically activeand diagnostically or therapeutically useful fragments, analogs andderivatives thereof.

[0014] In accordance with still another aspect of the present inventionthere are provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to hMLH1, hMLH2 and hMLH3sequences.

[0015] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptides byrecombinant techniques which comprises culturing recombinant prokaryoticand/or eukaryotic host cells, containing an hMLH1, hMLH2 or hMLH3nucleic acid sequence, under conditions promoting expression of saidprotein and subsequent recovery of said proteins.

[0016] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide, for therapeutic purposes, forexample, for the treatment of cancers.

[0017] In accordance with another aspect of the present invention thereis provided a method of diagnosing a disease or a susceptibility to adisease related to a mutation in the hMLH1, hMLH2 or hMLH3 nucleic acidsequences and the proteins encoded by such nucleic acid sequences.

[0018] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0019] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0021]FIG. 1 illustrates the cDNA sequence and corresponding deducedamino acid sequence for the human DNA repair protein hMLH1. The aminoacids are represented by their standard one-letter abbreviations.Sequencing was performed using a 373 Automated DNA sequencer (AppliedBiosystems, Inc.). Sequencing accuracy is predicted to be greater than97% accurate.

[0022]FIG. 2 illustrates the cDNA sequence and corresponding deducedamino acid sequence of hMLH2. The amino acids are represented by theirstandard one-letter abbreviations.

[0023]FIG. 3 illustrates the cDNA sequence and corresponding deducedamino acid sequence of hMLH3. The amino acids are represented by theirstandard one-letter abbreviations.

[0024]FIG. 4. Alignment of the predicted amino acid sequences of S.cerevisiae PMS1 (yPMS1), with the hMLH2 and hMLH3 amino acid sequencesusing MACAW (version 1.0) program. Amino acid in conserved blocks arecapitalized and shaded on the mean of their pair-wise scores.

[0025]FIG. 5. Mutational analysis of hMLH2. (A) IVSP analysis andmapping of the transcriptional stop mutation in HNPCC patient CW.Translation of codons 1 to 369 (lane 1), codons 1 to 290 (lane 2), andcodons 1 to 214 (lane 3). CW is translated from the cDNA of patient CW,while NOR was translated from the cDNA of a normal individual. Thearrowheads indicate the truncated polypeptide due to the potential stopmutation. The arrows indicate molecular weight markers in kilodaltons.(B) Sequence analysis of CW indicates a C to T transition at codon 233(indicated by the arrow). Lanes 1 and 3 are sequence derived fromcontrol patients; lane 2 is sequence derived from genomic DNA of CW. TheddA mixes from each sequencing mix were loaded in adjacent lanes tofacilitate comparison as were those for ddC, ddD, and ddT mixes.

[0026]FIG. 6. Mutational analysis of hMLH3. (A) IVSP analysis of hMLH3from patient GC. Lane GC is from fibroblasts of individual GC; lane GCxis from the tumor of patient GC; lanes NOR1 and 2 are from normalcontrol individuals. FL indicates full-length protein, and thearrowheads indicate the germ line truncated polypeptide. The arrowsindicate molecular weight markers in kilodaltons (B) PCR analysis of DNAfrom a patient GC shows that the lesion in present in both hMLH3 allelesin tumor cells. Amplification was done using primers that amplify 5′,3′, or within (MID) the region deleted in the cDNA. Lane 1, DNA derivedfrom fibroblasts of patient GC; lane 2, DNA derived from tumor ofpatient GC; lane 3, DNA derived from a normal control patient; lane 4,reactions without DNA template. Arrows indicate molecular weight in basepairs.

DETAILED DESCRIPTION OF THE INVENTION

[0027] In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptides having the deduced amino acid sequence of FIGS. 1, 2and 3 (SEQ ID NOS:2, 4 and 6) or for the mature polypeptides encoded bythe cDNA of the clone deposited as ATCC Deposit No. 75649, 75651, 75650,deposited on Jan. 25, 1994. The address of the American Type CultureCollection (ATCC) Depository referred to herein is: American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209.

[0028] ATCC Deposit No. 75649 is a cDNA clone which contains the fulllength sequence encoding the human DNA repair protein referred to hereinas hMLH1; ATCC Deposit No. 75651 is a cDNA clone containing the fulllength cDNA sequence encoding the human DNA repair protein referred toherein as hMLH2; ATCC Deposit No. 75650 is a cDNA clone containing thefull length DNA sequence referred to herein as hMLH3.

[0029] Polynucleotides encoding the polypeptides of the presentinvention may be obtained from one or more libraries prepared fromheart, lung, prostate, spleen, liver, gallbladder, fetal brain andtestes tissues. The polynucleotides of hMLH1 were discovered from ahuman gallbladder cDNA library. In addition, six cDNA clones which areidentical to the hMLH1 at the N-terminal ends were obtained from humancerebellum, eight-week embryo, fetal heart, HSC172 cells and Jurket cellcDNA libraries. The hMLH1 gene contains an open reading frame of 756amino acids encoding for an 85kD protein which exhibits homology to thebacterial and yeast mutL proteins. However, the 5′ non-translated regionwas obtained from the cDNA clone obtained from the fetal heart for thepurpose of extending the non-translated region to design theoligonucleotides.

[0030] The hMLH2 gene was derived from a human T-cell lymphoma cDNAlibrary. The hMLH2 cDNA clone identified an open reading frame of 2,796base pairs flanked on both sides by in-frame termination codons. It isstructurally related to the yeast PMS1 family. It contains an openreading frame encoding a protein of 932 amino acid residues. The proteinexhibits the highest degree of homology to yeast PMS1 with 27% identityand 82% similarity over the entire protein.

[0031] A second region of significant homology among the three PMSrelated proteins is in the carboxyl terminus, between codons 800 to 900.This region shares a 22% and 47% homology between yeast PMS1 protein andhMLH2 and hMLH3 proteins, respectively, while very little homology ofthis region was observed between these proteins, and the other yeastmutL homolog, yMLH1.

[0032] The hMLH3 gene was derived from a human endometrial tumor cDNAlibrary. The hMLH3 clone identified a 2,586 base pair open readingframe. It is structurally related to the yPMS2 protein family. Itcontains an open reading frame encoding a protein of 862 amino acidresidues. The protein exhibits the highest degree of homology to yPMS2with 32% identity and 66% similarity over the entire amino acidsequence.

[0033] It is significant with respect to a putative identification ofhMLH1, hMLH2 and hMLH3 that the GFRGEAL domain which is conserved inmutL homologs derived from E. coli is conserved in the amino acidsequences of, hMLH1, hMLH2 and hMLH3.

[0034] The polynucleotides of the present invention may be in the formof RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1, 2 and 3 (SEQ IDNO: 1) or that of the deposited clone or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptides asthe DNA of FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or the depositedcDNA(s).

[0035] The polynucleotides which encode for the mature polypeptides ofFIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or for the mature polypeptidesencoded by the deposited cDNAs may include: only the coding sequence forthe mature polypeptide; the coding sequence for the mature polypeptide(and optionally additional coding sequence) and non-coding sequence,such as introns or non-coding sequence 5′ and/or 3′ of the codingsequence for the mature polypeptide.

[0036] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0037] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptides having the deduced aminoacid sequences of FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or thepolypeptides encoded by the cDNA of the deposited clones. The variantsof the polynucleotides may be a naturally occurring allelic variant ofthe polynucleotides or a non-naturally occurring variant of thepolynucleotides.

[0038] Thus, the present invention includes polynucleotides encoding thesame mature polypeptides as shown in FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4and 6) or the same mature polypeptides encoded by the cDNA of thedeposited clones as well as variants of such polynucleotides whichvariants encode for a fragment, derivative or analog of the polypeptidesof FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or the polypeptides encodedby the cDNA of the deposited clones. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

[0039] As hereinabove indicated, the polynucleotides may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1, 2 and 3 (SEQ ID NO: 1, 3 and 5) or of thecoding sequence of the deposited clones. As known in the art, an allelicvariant is an alternate form of a polynucleotide sequence which may havea substitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

[0040] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptides of the present invention. The markersequence may be, for example, a hexa-histidine tag supplied by a pQE-9vector to provide for purification of the mature polypeptides fused tothe marker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host, e.g.COS-7 cells, is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767(1984)).

[0041] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0042] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0043] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1, 2 and 3 (SEQ IDNO: 1, 3 and 5) or the deposited cDNA(s).

[0044] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, 3 and 5 for example, forrecovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

[0045] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NOS:2, 4 and 6 as well as fragments thereof, which fragmentshave at least 30 bases and preferably at least 50 bases and topolypeptides encoded by such polynucleotides.

[0046] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0047] The present invention further relates to polypeptides which havethe deduced amino acid sequence of FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and6) or which have the amino acid sequence encoded by the depositedcDNA(s), as well as fragments, analogs and derivatives of suchpolypeptides.

[0048] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptides of FIGS. 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or thatencoded by the deposited cDNA(s), means polypeptides which retainessentially the same biological function or activity as suchpolypeptides. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

[0049] The polypeptides of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0050] The fragment, derivative or analog of the polypeptides of FIGS.1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or that encoded by the depositedcDNAs may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol). Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

[0051] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0052] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0053] The polypeptides of the present invention include the polypeptideof SEQ ID NOS:2, 4 and 6 (in particular the mature polypeptide) as wellas polypeptides which have at least 70% similarity (preferably at least70% identity) to the polypeptide of SEQ ID NOS:2, 4 and 6 and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of SEQ ID NOS:2, 4 and 6 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of SEQ ID NOS:2, 4 and 6 and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0054] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0055] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0056] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0057] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the hMLH1, hMLH2 and hMLH3 genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0058] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0059] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0060] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0061] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0062] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the proteins.

[0063] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0064] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen,Inc.), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any otherplasmid or vector may be used as long as they are replicable and viablein the host.

[0065] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and TRP. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0066] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0067] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0068] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0069] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0070] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), -factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

[0071] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0072] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0073] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0074] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0075] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0076] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0077] The polypeptides can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (BPLC) can be employed for finalpurification steps.

[0078] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated.

[0079] In accordance with a further aspect of the invention, there isprovided a process for determining susceptibility to cancer, inparticular, a hereditary cancer. Thus, a mutation in a human repairprotein, which is a human homolog of mutL, and in particular thosedescribed herein, indicates a susceptibility to cancer, and the nucleicacid sequences encoding such human homologs may be employed in an assayfor ascertaining such susceptibility. Thus, for example, the assay maybe employed to determine a mutation in a human DNA repair protein asherein described, such as a deletion, truncation, insertion, frameshift, etc., with such mutation being indicative of a susceptibility tocancer.

[0080] A mutation may be ascertained for example, by a DNA sequencingassay. Tissue samples, including but not limited to blood samples areobtained from a human patient. The samples are processed by methodsknown in the art to capture the RNA. First strand cDNA is synthesizedfrom the RNA samples by adding an oligonucleotide primer consisting ofpolythymidine residues which hybridize to the polyadenosine stretchpresent on the mRNA's. Reverse transcriptase and deoxynucleotides areadded to allow synthesis of the first strand cDNA. Primer sequences aresynthesized based on the DNA sequence of the DNA repair protein of theinvention. The primer sequence is generally comprised of 15 to 30 andpreferably from 18 to 25 consecutive bases of the human DNA repair gene.Table 1 sets forth an illustrative example of oligonucleotide primersequences based on hMLH1. The primers are used in pairs (one “sense”strand and one “anti-sense”) to amplify the cDNA from the patients bythe PCR method (Saiki et al., Nature, 324:163-166 (1986)) such thatthree overlapping fragments of the patient's cDNA's for such protein aregenerated. Table 1 also shows a list of preferred primer sequence pairs.The overlapping fragments are then subjected to dideoxynucleotidesequencing using a set of primer hesized to correspond to the base pairsof the cDNA's at a point every 200 base pairs throughout the gene. TABLE1 Primer Sequences used to amplify gene region using PCR SEQ ID StartSite Name NO: and Arrangement Sequence 758 7 sense-(-41)*GTTGAACATCTAGACGTCTC 1319 8 sense-8 TCGTGGCAGGGGTTATTCG 1321 9 sense-619CTACCCAATGCCTCAACCG 1322 10 sense-677 GAGAACTGATAGAAATTGGATG 1314 11sense-1548 GGGACATGAGGTTCTCCG 1323 12 sense-1593 GGGCTGTGTGAATCCTCAG 77313 anti-53 CGGTTCACCACTGTCTCGTC 1313 14 anti-971 TCCAGGATGCTCTCCTCG 132015 anti-1057 CAAGTCCTGGTAGCAAAGTC 1315 16 anti-1760 ATGGCAAGGTCAAAGAGCG1316 17 anti-1837 CAACAATGTATTCAGXAAGTCC 1317 18 anti-2340TTGATACAACACTTTGTATCG 1318 19 anti-2415 GGAATACTATCAGAAGGCAAG

[0081] Preferred primer sequences pairs:

[0082] 758, 1313

[0083] 1319, 1320

[0084] 660, 1909

[0085] 725, 1995

[0086] 1680, 2536

[0087] 1727, 2610

[0088] The nucleotide sequences shown in Table 1 represent SEQ ID NO:7through 19, respectively.

[0089] Table 2 lists representative examples of oligonucleotide primersequences (sense and anti-sense) which may be used, and preferably theentire set of primer sequences are used for sequencing to determinewhere a mutation in the patient DNA repair protein may be. The primersequences may be from 15 to 30 bases in length and are preferablybetween 18 and 25 bases in length. The sequence information determinedfrom the patient is then compared to non-mutated sequences to determineif any mutations are present. TABLE 2 Primer Sequences Used to Sequencethe Amplified Fragments SEQ ID Start Site Name NO: and ArrangementSequence 5282 20 sense-377* ACAGAGCAAGTTACTCAGATG 5283 21 sense-552GTACACAATGCAGGCATTAG 5284 22 sense-904 AATGTGGATGTTAATGTGCAC 5285 23sense-1096 CTGACCTCGTGTTCCTAC 5286 24 sense-1276 CAGCAAGATGAGGAGATGC5287 25 sense-1437 GGAAATGGTGGAAGATGATTC 5288 26 sense-1645CTTCTCAACACCAAGC 5289 27 sense-1895 GAAATTGATGAGGAAGGGAAC 5295 28sense-1921 CTTCTGATTGACAACTATGTGC 5294 29 sense-2202CACAGAAGATGGAAATATCCTG 293 30 sense-2370 GTGTTGGTAGCACTTAAGAC 5291 31anti-525 TTTCCCATATTCTTCACTTG 5290 32 anti-341 GTAACATGAGCCACATGGC 529233 anti-46 CCACTGTCTCGTCCAGCCG

[0090] The nucleotide sequences shown in Table 2 represent SEQ ID NO:20through 33, respectively.

[0091] In another embodiment, the primer sequences from Table 2 could beused in the PCR method to amplify a mutated region. The region could besequenced and used as a diagnostic to predict a predisposition to suchmutated genes.

[0092] Alternatively, the assay to detect mutations in the genes of thepresent invention may be performed by genetic testing based on DNAsequence differences achieved by detection of alteration inelectrophoretic mobility of DNA fragments in gels with or withoutdenaturing agents. Small sequence deletions and insertions can bevisualized by high resolution gel electrophoresis. DNA fragments ofdifferent sequences may be distinguished on denaturing formamidegradient gels in which the mobilities of different DNA fragments areretarded in the gel at different positions according to their specificmelting or partial melting temperatures (see, e.g., Myers et al.,Science, 230:1242 (1985)).

[0093] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)). Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0094] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,Western Blot analysis, direct DNA sequencing or the use of restrictionenzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) andSouthern blotting of genomic DNA.

[0095] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0096] The polypeptides may also be employed to treat cancers or toprevent cancers, by expression of such polypeptides in vivo, which isoften referred to as “gene therapy.”

[0097] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0098] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0099] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0100] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and -actin promoters). Other viral promoters which maybe employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0101] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the -actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0102] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, -2, -AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0103] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0104] Each of the cDNA sequences identified herein or a portion thereofcan be used in numerous ways as polynucleotide reagents. The sequencescan be used as diagnostic probes for the presence of a specific mRNA ina particular cell type. In addition, these sequences can be used asdiagnostic probes suitable for use in genetic linkage analysis(polymorphisms).

[0105] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0106] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0107] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome-specific cDNAlibraries.

[0108] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0109] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0110] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0111] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0112] hMLH2 has been localized using a genomic P1 clone (1670) whichcontained the 5′ region of the hMLH2 gene. Detailed analysis of humanmetaphase chromosome spreads, counterstained to reveal banding,indicated that the hMLH2 gene was located within bands 2q32. Likewise,hMLH3 was localized using a genomic P1 clone (2053) which contained the3′ region of the hMLH3 gene. Detailed analysis of human metaphasechromosome spreads, counterstained to reveal banding, indicated that thehMLH3 gene was located within band 7p22, the most distal band onchromosome 7. Analysis with a variety of genomic clones showed thathMLH3 was a member of a subfamily of related genes, all on chromosome 7.

[0113] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0114] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0115] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0116] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0117] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0118] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0119] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0120] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0121] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0122] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0123] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0124] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLES Example 1 Bacterial Expression of hMLH1

[0125] The full length DNA sequence encoding human DNA mismatch repairprotein hMLH1, ATCC #75649, is initially amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ ends of the DNAsequence to synthesize insertion fragments. The 5′ oligonucleotideprimer has the sequence 5′ CGGGATCCATGTCGTTCGTGGCAGGG 3′ (SEQ ID NO:34),contains a BamHI restriction enzyme site followed by 18 nucleotides ofhMLH1 coding sequence following the initiation codon; the 3′ sequence 5′GCTCTAGATTAACACCTCTCAAAGAC 3′ (SEQ ID NO:35) contains complementarysequences to an XbaI site and is at the end of the gene. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). The plasmidvector encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter/operator (P/O), aribosome binding site (RBS), a 6-histidine tag (6-His) and restrictionenzyme cloning sites. The pQE-9 vector is digested with BamHI and XbaIand the insertion fragments are then ligated into the pQE-9 vectormaintaining the reading frame initiated at the bacterial RBS. Theligation mixture is then used to transform the E. coli strain M15/rep4(Qiagen, Inc.) which contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). Tho O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is thenadded to a final concentration of 1 mM. IPTG induces by inactivating thelacI repressor, clearing the P/O leading to increased gene expression.Cells are grown an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000× g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized hMLH1 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., GeneticEngineering, Principles & Methods, 12:87-98 (1990). Protein renaturationout of GnHCl can be accomplished by several protocols (Jaenicke, R. andRudolph, R., Protein Structure—A Practical Approach, IRL Press, New York(1990)). Initially, step dialysis is utilized to remove the GnHCL.Alternatively, the purified protein isolated from the Ni-chelate columncan be bound to a second column over which a decreasing linear GnHCLgradient is run. The protein is allowed to renature while bound to thecolumn and is subsequently eluted with a buffer containing 250 mMImidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally,soluble protein is dialyzed against a storage buffer containing 5 mMAmmonium Bicarbonate. The purified protein was analyzed by SDS-PAGE.

Example 2 Spontaneous Mutation Assay for Detection of the Expression ofhMLH1, hMLH2 and hMLH3 and Complementation to the E.coli mutl

[0126] The pQE9hMLH1, pQE9hMLH2 or pQE9hMLH3/GW3733, transformants weresubjected to the spontaneous mutation assay. The plasmid vector pQE9 wasalso transformed to AB1157 (k-12, argE3 hisG4,LeuB6 proA2 thr-1 ara-1rpsL31 supE44 tsx-33) and GW3733 to use as the positive and negativecontrol respectively.

[0127] Fifteen 2 ml cultures, inoculated with approximately 100 to 1000E. coli, were grown 2×10⁸ cells per ml in LB ampicillin medium at 37° C.Ten microliters of each culture were diluted and plated on the LBampicillin plates to measure the number of viable cells. The rest of thecells from each culture were then concentrated in saline and plated onminimal plates lacking of arginine to measure reversion of Arg⁺. InTable 3, the mean number of mutations per culture (m) was calculatedfrom the median number (r) of mutants per distribution, according to theequation (r/m)-ln(m)=1.24 (Lea et al., J. Genetics 49:264-285 (1949)).Mutation rates per generation were recorded as m/N, with N representingthe average number of cells per culture. TABLE 3 Spontaneous MutationRates Strain Mutation/generation AB1157 + vector (5.6 ± 0.1) × 10 − 9aGW3733 + vector (1.1 ± 0.2) × 10 − 6a GW3733 + phMLH1 (3.7 ± 1.3) × 10 −7a GW3733 + phMLH2 (3.1 ± 0.6) × 10 − 7b GW3733 + phMLH3 (2.1 ± 0.8) ×10 − 7b

[0128] The functional complementation result showed that the human mutLcan partially rescue the E.coli mutL mutator phenotype, suggesting thatthe human mutL is not only successfully expressed in a bacterialexpression system, but also functions in bacteria.

Example 3 Chromosomal Mapping of the hMLH1

[0129] An oligonucleotide primer set was designed according to thesequence at the 5′ end of the cDNA for HMLH1. This primer set would spana 94 bp segment. This primer set was used in a polymerase chain reactionunder the following set of conditions:

[0130] 30 seconds, 95 degrees C.

[0131] 1 minute, 56 degrees C.

[0132] 1 minute, 70 degrees C.

[0133] This cycle was repeated 32 times followed by one 5 minute cycleat 70 degrees C. Human, mouse, and hamster DNA were used as template inaddition to a somatic cell hybrid panel (Bios, Inc). The reactions wereanalyzed on either 8% polyacrylamide gels or 3.5% agarose gels. A 94base pair band was observed in the human genomic DNA sample and in thesomatic cell hybrid sample corresponding to chromosome 3. In addition,using various other somatic cell hybrid genomic DNA, the hMLH1 gene waslocalized to chromosome 3p.

Example 4 Method for Determination of Mutation of hMLH1 Gene in HNPCCKindred

[0134] cDNA was produced from RNA obtained from tissue samples frompersons who are HNPCC kindred and the cDNA was used as a template forPCR, employing the primers 5′ GCATCTAGACGTTTCCTTGGC 3′ (SEQ ID NO:36)and 5′ CATCCAAGCTTCTGTTCCCG 3′ (SEQ ID NO:37), allowing amplification ofcodons 1 to 394 of FIG. 1; 5′ GGGGTGCAGCAGCACATCG 3′ (SEQ ID NO:38) and5′ GGAGGCAGAATGTGTGAGCG 3′ (SEQ ID NO:39), allowing amplification ofcodons 326 to 729 of FIG. 1 (SEQ ID NO:2); and 5′ TCCCAAAGAAGGACTTGCT 3′(SEQ ID NO:40) and 5′ AGTATAAGTCTTAAGTGCTACC 3′ (SEQ ID NO:41), allowingamplification of codons 602 to 756 plus 128 nt of 3′-untranslatedsequences of FIG. 1 (SEQ ID NO:2). The PCR conditions for all analysesused consisted of 35 cycles at 95 C for 30 seconds, 52-58 C for 60 to120 seconds, and 70 C for 60 to 120 seconds, in the buffer solutiondescribed in San Sidransky, D. et al., Science, 252:706 (1991). PCRproducts were sequenced using primers labeled at their 5′ end with T4polynucleotide kinase, employing SequiTherm Polymerase (EpicentreTechnologies). The intron-exon borders of selected exons were alsodetermined and genomic PCR products analyzed to confirm the results. PCRproducts harboring suspected mutations were then cloned and sequenced tovalidate the results of the direct sequencing. PCR products were clonedinto T-tailed vectors as described in Holton, T. A. and Graham, M. W.,Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase(United States Biochemical). Affected individuals from seven kindredsall exhibited a heterozygous deletion of codons 578 to 632 of the hMLH1gene. The derivation of five of these seven kindreds could be traced toa common ancestor. The genomic sequences surrounding codons 578-632 weredetermined by cycle-sequencing of the P1 clones (a human genomic P1library which contains the entire hMLH1 gene (Genome Systems)) usingSequiTherm Polymerase, as described by the manufacturer, with theprimers were labeled with T4 polynucleotide kinase, and by sequencingPCR products of genomic DNA. The primers used to amplify the exoncontaining codons 578-632 were 5′ TTTATGGTTTCTCACCTGCC 3′ (SEQ ID NO:42)and 5′ GTTATCTGCCCACCTCAGC 3′ (SEQ ID NO:43). The PCR product included105 bp of intron C sequence upstream of the exon and 117 bp downstream.No mutations in the PCR product were observed in the kindreds, so thedeletion in the RNA was not due to a simple splice site mutation. Codons578 to 632 were found to constitute a single exon which was deleted fromthe gene product in the kindreds described above. This exon containsseveral highly conserved amino acids.

[0135] In a second family (L7), PCR was performed using the aboveprimers and a 4 bp deletion was observed beginning at the firstnucleotide (nt) of codon 727. This produced a frame shift with a newstop codon 166 nt downstream, resulting in a substitution of thecarboxy-terminal 29 amino acids of hMLH1 with 53 different amino acids,some encoded by nt normally in the 3′ untranslated region.

[0136] A different mutation was found in a different kindred (L2516)after PCR using the above primers, the mutation consisting of a 4 bpinsert between codons 755 and 756. This insertion resulted in a frameshift and extension of the ORF to include 102 nucleotides (34 aminoacids) downstream of the normal termination codon. The mutations in bothkindreds L7 and L2516 were therefore predicted to alter the C-terminusof hMLH1.

[0137] A possible mutation in the hMLH1 gene was determined fromalterations in size of the encoded protein, where kindreds were too fewfor linkage studies. The primers used for coupledtranscription-translation of hMLH1 were 5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGCATCTAGACGTTT CCCTTGGC 3′ (SEQ IDNO:44) and 5′ CATCCAAGCTTCTGTTCCCG 3′ (SEQ ID NO:45) for codons 1 to 394of FIG. 1 and 5′ GGATCCTAATACGACTCACTATAGGGAGACCACCATGGGGGTGCAGCAGCACATCG 3′ (SEQ ID NO:46) and 5′ GGAGGCAGAATGTGTGAGCG 3′ (SEQ ID NO:47)for codons 326 to 729 of FIG. 1 (SEQ ID NO:2). The resultant PCRproducts had signals for transcription by T7 RNA polymerase and for theinitiation of translation at their 5′ ends. RNA from lymphoblastoidcells of patients from 18 kindreds was used to amplify two products,extending from codon 1 to codon 394 or from codon 326 to codon 729,respectively. The PCR products were then transcribed and translated invitro, making use of transcription-translation signals incorporated intothe PCR primers. PCR products were used as templates in coupledtranscription-translation reactions performed as described by Powell, S.M. et al., New England Journal of Medicine, 329:1982, (1993), using 40micro CI of ³⁵S labeled methionine. Samples were diluted in samplebuffer, boiled for five minutes and analyzed by electropheresis onsodium dodecyl sulfate-polyacrylamide gels containing a gradient of 10%to 20% acrylamide. The gels were dried and subjected to radiography. Allsamples exhibited a polypeptide of the expected size, but an abnormallymigrating polypeptide was additionally found in one case. The sequenceof the relevant PCR product was determined and found to include a 371 bpdeletion beginning at the first nucleotide (nt) of codon 347. Thisalteration was present in heterozygous form, and resulted in a frameshift in a new stop codon 30 nt downstream of codon 346, thus explainingthe truncated polypeptide observed.

[0138] Four colorectal tumor cell lines manifesting microsatelliteinstability were examined. One of the four (cell line H6) showed nonormal peptide in this assay and produced only a short product migratingat 27 kd. The sequence of the corresponding cDNA was determined andfound to harbor a C to A transversion at codon 252, resulting in thesubstitution of a termination codon for serine. In accord with thetranslational analyses, no band at the normal C position was identifiedin the cDNA or genomic DNA from this tumor, indicating that it wasdevoid of a functional hMLH1 gene.

[0139] Table 4 sets forth the results of these sequencing assays.Deletions were found in those people who were known to have a familyhistory of the colorectal cancer. More particularly, 9 of 10 familiesshowed an hMLH1 mutation. TABLE 4 Summary of Mutations in hMLH1 cDNANucleotide Predicted Sample Codon Change Coding Change Kindreds F2, F3,F6, 578-632 165 bp deletion In-frame deletion E8, F10, F11, F52 KindredL7 727/728 4 bp deletion Frameshift and (TCACACATTC substitution of newamino to TCATTCT) acids Kindred L2516 755/756 4 bp insertion Extensionof C-terminus (GTGTTAA to GTGTTTGTTAA) Kindred RA 347 371 bp deletionFrameshift/Truncation H6 Colorectal Tumor 252 Transversion Serine toStop (TCA to TAA)

Example 5 Bacterial Expression and Purification of hMLH2

[0140] The DNA sequence encoding hMLH2, ATCC #75651, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence to synthesize insertion fragments. The 5′oligonucleotide primer has the sequence 5′ CGGGATCCATGAAACAATTGCCTGCGGC3′ (SEQ ID NO:48) contains a BamHI restriction enzyme site followed by17 nucleotides of hMLH2 following the initiation codon. The 3′ sequence5′ GCTCTAGACCAGACTCATGCTGTTTT 3′ (SEQ ID NO:49) contains complementarysequences to an XbaI site and is followed by 18 nucleotides of hMLH2.The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth,Calif.). pQE-9 encodes antibiotic resistance (Amp^(r)), a bacterialorigin of replication (ori), an IPTG-regulatable promoter operator(P/O), a ribosome binding site (RBS), a 6-His tag and restriction enzymesites. The amplified sequences and pQE-9 are then digested with BamHIand XbaI. The amplified sequences are ligated into pQE-9 and areinserted in frame with the sequence encoding for the histidine tag andthe RBS. The ligation mixture is then used to transform E. coli strainM15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmidpREP4, which expresses the lacI repressor and also confers kanamycinresistance (Kan^(r)). Transformants are identified by their ability togrow on LB plates and ampicillin/kanamycin resistant colonies areselected. Plasmid DNA is isolated and confirmed by restriction analysis.Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). Tho O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells are grown an extra 3 to 4 hours. Cellsare then harvested by centrifugation (20 mins at 6000× g). The cellpellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl.After clarification, solubilized hMLH2 is purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., Genetic Engineering, Principles & Methods, 12:87-98 (1990). Proteinrenaturation out of GnHCl can be accomplished by several protocols(Jaenicke, R. and Rudolph, R., Protein Structure—A Practical Approach,IRL Press, New York (1990)). Initially, step dialysis is utilized toremove the GnHCL. Alternatively, the purified protein isolated from theNi-chelate column can be bound to a second column over which adecreasing linear GnHCL gradient is run. The protein is allowed torenature while bound to the column and is subsequently eluted with abuffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5and 10% Glycerol. Finally, soluble protein is dialyzed against a storagebuffer containing 5 mM Ammonium Bicarbonate. The purified protein wasanalyzed by SDS-PAGE.

Example 6 Bacterial Expression and Purification of hMLH3

[0141] The DNA sequence encoding hMLH3, ATCC #75650, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence to synthesize insertion fragments. The 5′oligonucleotide primer has the sequence 5′ CGGGATCCATGGAGCGAGCTGAGAGC 3′(SEQ ID NO:50) contains a BamHI restriction enzyme site followed by 18nucleotides of hMLH3 coding sequence starting from the presumed terminalamino acid of the processed protein. The 3′ sequence 5′ GCTCTAGAGTGAAG.

[0142] ACTCTGTCT 3′ (SEQ ID NO:51) contains complementary sequences toan XbaI site and is followed by 18 nucleotides of hMLH3. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter operator (P/O), a ribosome bindingsite (RBS), a 6-His tag and restriction enzyme sites. The amplifiedsequences and pQE-9 are then digested with BamHI and XbaI. The amplifiedsequences are ligated into pQE-9 and are inserted in frame with thesequence encoding for the histidine tag and the RBS. The ligationmixture was then used to transform E. coli strain M15/rep4 (Qiagen,Inc.) which contains multiple copies of the plasmid pREP4, whichexpresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). Tho O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is thenadded to a final concentration of 1 mM. IPTG induces by inactivating thelacI repressor, clearing the P/O leading to increased gene expression.Cells are grown an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000× g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized stanniocalcin is purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., Genetic Engineering, Principles & Methods, 12:87-98 (1990). Proteinrenaturation out of GnHCl can be accomplished by several protocols(Jaenicke, R. and Rudolph, R., Protein Structure—A Practical Approach,IRL Press, New York (1990)). Initially, step dialysis is utilized toremove the GnHCL. Alternatively, the purified protein isolated from theNi-chelate column can be bound to a second column over which adecreasing linear GnHCL gradient is run. The protein is allowed torenature while bound to the column and is subsequently eluted with abuffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5and 10% Glycerol. Finally, soluble protein is dialyzed against a storagebuffer containing 5 mM Ammonium Bicarbonate. The purified protein wasanalyzed by SDS-PAGE.

Example 7 Method for Determination of Mutation of hMLH2 and hMLH3 inHereditary Cancer

[0143] Isolation of Genomic Clones

[0144] A human genomic P1 library (Genomic Systems, Inc.) was screenedby PCR using primers selected for the cDNA sequence of hMLH2 and hMLH3.Two clones were isolated for hMLH2 using primers 5′ AAGCTGCTCTGTTAAAAGCG3′ (SEQ ID NO:52) and 5′ GCACCAGCATCCAAGGAG 3′ (SEQ ID NO:53) andresulting in a 133 bp product. Three clones were isolated for hMLH3,using primers 5′ CAACCATGAGACACATCGC 3′ (SEQ ID NO:54) and 5′AGGTTAGTGAAGACTCTGTC 3′ (SEQ ID NO:55) resulting in a 121 bp product.Genomic clones were nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH was performed asdescribed (Johnson, Cg. et al., Methods Cell Biol., 35:73-99 (1991)).Hybridization with the hMLH3 probe were carried out using a vast excessof human cot-1 DNA for specific hybridization to the expressed hMLH3locus. Chromosomes were counterstained with 4,6-diamino-2-phenylidoleandpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping were obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991)). Image collection, analysis and chromosomalfractional length measurements were done suing the ISee GraphicalProgram System (Inovision Corporation, Durham, N.C.).

[0145] Transcription Coupled Translation Mutation Analysis

[0146] For purposes of IVSP analysis the hMLH2 gene was divided intothree overlapping segments. The first segment included codons 1 to 500,while the middle segment included codons 270 to 755, and the lastsegment included codons 485 to the translational termination site atcodon 933. The primers for the first segment were 5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAACAATTGCCTGC GG 3′ (SEQ ID NO:56)and 5′ CCTGCTCCACTCATCTGC 3′ (SEQ ID NO:57), for the middle segment were5′ GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAAGATATCTTAAA GTTAATCCG 3′ (SEQID NO:58) and 5′ GGCTTCTTCTACTCTATATGG 3′ (SEQ ID NO:59), and for thefinal segment were 5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGCAGGTCTTGAAAA CTCTTCG 3′ (SEQ IDNO:60) and 5′ AAAACAAGTCAGTGAATCCTC 3′ (SEQ ID NO:61). The primers usedfor mapping the stop mutation in patient CW all used the same 5′ primeras the first segment. The 3′ nested primers were: 5′AAGCACATCTGTTTCTGCTG 3′ (SEQ ID NO:62) codons 1 to 369; 5′ACGAGTAGATTCCTTTAGGC 3′ (SEQ ID NO:63) codons 1 to 290; and 5′CAGAACTGACATGAGAGCC 3′ (SEQ ID NO:64) codons 1 to 214.

[0147] For analysis of hMLH3, the hMLH3 cDNA was amplified as afull-length product or as two overlapping segments. The primers forfull-length hMLH3 were 5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAGCGAGCTGAGAG C 3′ (SEQ ID NO:65)and 5′ AGGTTAGTGAAGACTCTGTC 3′ (SEQ ID NO:66) (codons 1 to 863). Forsegment 1, the sense primer was the same as above and the antisenseprimer was 5′ CTGAGGTCTCAGCAGGC 3′ (SEQ ID NO:67) (codons 1 to 472).Segment 2 primers were 5′GGATCCTAATACGACTCACTATAGGGAGACCACCATGGTGTCCATTTCCAG ACTGCG 3′ (SEQ IDNO:68) and 5′ AGGTTAGTGAAGACTCTGTC 3′ (SEQ ID NO:69) (codons 415 to863). Amplifications were done as described below.

[0148] The PCR products contained recognition signals for transcriptionby T7 RNA polymerase and for the initiation of translation at thei 5′ends. PCR products were used as templates in coupledtranscription-translation reactions containing 40 uCi of ³⁶S-methionine(NEN, Dupont). Samples were diluted in SDS sample buffer, and analyzedby electrophoresis on SDS-polyacrylamide gels containing a gradient of10 to 20% acrylamide. The gels were fixed, treated with EnHance(Dupont), dried and subjected to autoradiography.

[0149] RT-PCR and Direct Sequencing of PCR Products

[0150] cDNAs were generated from RNA of lymphoblastoid or tumor cellswith Superscript II (Life Technologies). The cDNAs were then used astemplates for PCR. The conditions for all amplifications were 35 cyclesat 95 C for 30s, 52 C to 62 C for 60 to 120s, and 70 C for 60 to 120s,in buffer. The PCR products were directly sequenced and cloned into theT-tailed cloning vector PCR2000 (Invitrogen) and sequenced with T7polymerase (United States Biochemical). For the direct sequencing of PCRproducts, PCR reactions were first phenolchloroform extracted andethanol precipitated. Templates were directly sequenced using Sequithermpolymerase (Epicentre Technologies) and gamma-³²p labelled primers asdescribed by the manufacturer. Intron/Exon Boundaries and GenomicAnalysis of Mutations

[0151] Intron/exon borders were determined by cycle-sequencing P1 clonesusing gamma-³²P end labelled primers and SequiTherm polymerase asdescribed by the manufacturer. The primers used to amplify the hMLH2exon containing codons 195 to 233 were 5′ TTATTTGGCAGAAAAGCAGAG (SEQ IDNO:70) 3′ and 5′ TTAAAAGACTAACCTCTTGCC 3′ (SEQ ID NO:71), which produceda 215 bp product. The product was cycle sequenced using the primer 5′CTGCTGTTATGAACAATATGG 3′ (SEQ ID NO:72). The primers used to analyze thegenomic deletion of hMLH3 in patient GC were: for the 5′ regionamplification 5′ CAGAAGCAGTTGCAAAGCC 3′ (SEQ ID NO:73) and 5′AAACCGTACTCTTCACACAC 3′ (SEQ ID NO:74) which produces a 74 bp productcontaining codons 233 to 257, primers 5′ GAGGAAAAGCTTTTGTTGGC 3′ (SEQ IDNO:75) and 5′ CAGTGGCTGCTGACTGAC 3′ (SEQ ID NO:76) which produce a 93 bpproduct containing the codons 347 to 377, and primers 5′TCCAGAACCAAGAAGGAGC 3′ (SEQ ID NO:77) and 5′ TGAGGTCTCAGCAGGC 3′ (SEQ IDNO:78) which produce a 99 bp product containing the codons 439 to 472 ofhMLH3. TABLE 5 Summary of Mutations in HMLH2 and HMLH3 from patientsaffected with HNPCC Nucleo- cDNA Genomic Predicted Sample Codon tidesChange Change Coding Change HMLH2 CW 233 Skipped CAG to GLN to Stop ExonTAG Codon HMLH3 MM, NS, 20 CGG CGG to ARG to GLN TF CAG CAG GC 268 to1,203 bp Deletion In frame 669 Deletion deletion GCx 268 to 1,203 bpDeletion Frameshift, 669 Deletion Truncation

Example 8 Expression via Gene Therapy

[0152] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37 C forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0153] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0154] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0155] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0156] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirally all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0157] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0158] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 78 1 2525 DNA homo sapiens CDS (42)..(2312) 1 gttgaacatc tagacgtttccttggctctt ctggcgccaa a atg tcg ttc gtg gca 56 Met Ser Phe Val Ala 1 5ggg gtt att cgg cgg ctg gac gag aca gtg gtg aac cgc atc gcg gcg 104 GlyVal Ile Arg Arg Leu Asp Glu Thr Val Val Asn Arg Ile Ala Ala 10 15 20 ggggaa gtt atc cag cgg cca gct aat gct atc aaa gag atg att gag 152 Gly GluVal Ile Gln Arg Pro Ala Asn Ala Ile Lys Glu Met Ile Glu 25 30 35 aac tgttta gat gca aaa tcc aca agt att caa gtg att gtt aaa gag 200 Asn Cys LeuAsp Ala Lys Ser Thr Ser Ile Gln Val Ile Val Lys Glu 40 45 50 gga ggc ctgaag ttg att cag atc caa gac aat ggc acc ggg atc agg 248 Gly Gly Leu LysLeu Ile Gln Ile Gln Asp Asn Gly Thr Gly Ile Arg 55 60 65 aaa gaa gat ctggat att gta tgt gaa agg ttc act act agt aaa ctg 296 Lys Glu Asp Leu AspIle Val Cys Glu Arg Phe Thr Thr Ser Lys Leu 70 75 80 85 cag tcc ttt gaggat tta gcc agt att tct acc tat ggc ttt cga ggt 344 Gln Ser Phe Glu AspLeu Ala Ser Ile Ser Thr Tyr Gly Phe Arg Gly 90 95 100 gag gct ttg gccagc ata agc cat gtg gct cat gtt act att aca acg 392 Glu Ala Leu Ala SerIle Ser His Val Ala His Val Thr Ile Thr Thr 105 110 115 aaa aca gct gatgga aag tgt gca tac aga gca agt tac tca gat gga 440 Lys Thr Ala Asp GlyLys Cys Ala Tyr Arg Ala Ser Tyr Ser Asp Gly 120 125 130 aaa ctg aaa gcccct cct aaa cca tgt gct ggc aat caa ggg acc cag 488 Lys Leu Lys Ala ProPro Lys Pro Cys Ala Gly Asn Gln Gly Thr Gln 135 140 145 atc acg gtg gaggac ctt ttt tac aac ata gcc acg agg aga aaa gct 536 Ile Thr Val Glu AspLeu Phe Tyr Asn Ile Ala Thr Arg Arg Lys Ala 150 155 160 165 tta aaa aatcca agt gaa gaa tat ggg aaa att ttg gaa gtt gtt ggc 584 Leu Lys Asn ProSer Glu Glu Tyr Gly Lys Ile Leu Glu Val Val Gly 170 175 180 agg tat tcagta cac aat gca ggc att agt ttc tca gtt aaa aaa caa 632 Arg Tyr Ser ValHis Asn Ala Gly Ile Ser Phe Ser Val Lys Lys Gln 185 190 195 gga gag acagta gct gat gtt agg aca cta ccc aat gcc tca acc gtg 680 Gly Glu Thr ValAla Asp Val Arg Thr Leu Pro Asn Ala Ser Thr Val 200 205 210 gac aat attcgc tcc gtc ttt gga aat gct gtt agt cga gaa ctg ata 728 Asp Asn Ile ArgSer Val Phe Gly Asn Ala Val Ser Arg Glu Leu Ile 215 220 225 gaa att ggatgt gag gat aaa acc cta gcc ttc aaa atg aat ggt tac 776 Glu Ile Gly CysGlu Asp Lys Thr Leu Ala Phe Lys Met Asn Gly Tyr 230 235 240 245 ata tccaat gca aac tac tca gtg aag aag tgc atc ttc tta ctc ttc 824 Ile Ser AsnAla Asn Tyr Ser Val Lys Lys Cys Ile Phe Leu Leu Phe 250 255 260 atc aaccat cgt ctg gta gaa tca act tcc ttg aga aaa gcc ata gaa 872 Ile Asn HisArg Leu Val Glu Ser Thr Ser Leu Arg Lys Ala Ile Glu 265 270 275 aca gtgtat gca gcc tat ttg ccc aaa aac aca cac cca ttc ctg tac 920 Thr Val TyrAla Ala Tyr Leu Pro Lys Asn Thr His Pro Phe Leu Tyr 280 285 290 ctc agttta gaa atc agt ccc cag aat gtg gat gtt aat gtg cac ccc 968 Leu Ser LeuGlu Ile Ser Pro Gln Asn Val Asp Val Asn Val His Pro 295 300 305 aca aagcat gaa gtt cac ttc ctg cac gag gag agc atc ctg gag cgg 1016 Thr Lys HisGlu Val His Phe Leu His Glu Glu Ser Ile Leu Glu Arg 310 315 320 325 gtgcag cag cac atc gag agc aag ctc ctg ggc tcc aat tcc tcc agg 1064 Val GlnGln His Ile Glu Ser Lys Leu Leu Gly Ser Asn Ser Ser Arg 330 335 340 atgtac ttc acc cag act ttg cta cca gga ctt gct ggc ccc tct ggg 1112 Met TyrPhe Thr Gln Thr Leu Leu Pro Gly Leu Ala Gly Pro Ser Gly 345 350 355 gagatg gtt aaa tcc aca aca agt ctg acc tcg tct tct act tct gga 1160 Glu MetVal Lys Ser Thr Thr Ser Leu Thr Ser Ser Ser Thr Ser Gly 360 365 370 agtagt gat aag gtc tat gcc cac cag atg gtt cgt aca gat tcc cgg 1208 Ser SerAsp Lys Val Tyr Ala His Gln Met Val Arg Thr Asp Ser Arg 375 380 385 gaacag aag ctt gat gca ttt ctg cag cct ctg agc aaa ccc ctg tcc 1256 Glu GlnLys Leu Asp Ala Phe Leu Gln Pro Leu Ser Lys Pro Leu Ser 390 395 400 405agt cag ccc cag gcc att gtc aca gag gat aag aca gat att tct agt 1304 SerGln Pro Gln Ala Ile Val Thr Glu Asp Lys Thr Asp Ile Ser Ser 410 415 420ggc agg gct agg cag caa gat gag gag atg ctt gaa ctc cca gcc cct 1352 GlyArg Ala Arg Gln Gln Asp Glu Glu Met Leu Glu Leu Pro Ala Pro 425 430 435gct gaa gtg gct gcc aaa aat cag agc ttg gag ggg gat aca aca aag 1400 AlaGlu Val Ala Ala Lys Asn Gln Ser Leu Glu Gly Asp Thr Thr Lys 440 445 450ggg act tca gaa atg tca gag aag aga gga cct act tcc agc aac ccc 1448 GlyThr Ser Glu Met Ser Glu Lys Arg Gly Pro Thr Ser Ser Asn Pro 455 460 465aga aag aga cat cgg gaa gat tct gat gtg gaa atg gtg gaa gat gat 1496 ArgLys Arg His Arg Glu Asp Ser Asp Val Glu Met Val Glu Asp Asp 470 475 480485 tcc cga aag gaa atg act gca gct tgt acc ccc cgg aga agg atc att 1544Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro Arg Arg Arg Ile Ile 490 495500 aac ctc act agt gtt ttg agt ctc cag gaa gaa att aat gag cag gga 1592Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu Ile Asn Glu Gln Gly 505 510515 cat gag gtt ctc cgg gag atg ttg cat aac cac tcc ttc gtg ggc tgt 1640His Glu Val Leu Arg Glu Met Leu His Asn His Ser Phe Val Gly Cys 520 525530 gtg aat cct cag tgg gcc ttg gca cag cat caa acc aag tta tac ctt 1688Val Asn Pro Gln Trp Ala Leu Ala Gln His Gln Thr Lys Leu Tyr Leu 535 540545 ctc aac acc acc aag ctt agt gaa gaa ctg ttc tac cag ata ctc att 1736Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe Tyr Gln Ile Leu Ile 550 555560 565 tat gat ttt gcc aat ttt ggt gtt ctc agg tta tcg gag cca gca ccg1784 Tyr Asp Phe Ala Asn Phe Gly Val Leu Arg Leu Ser Glu Pro Ala Pro 570575 580 ctc ttt gac ctt gcc atg ctt gcc tta gat agt cca gag agt ggc tgg1832 Leu Phe Asp Leu Ala Met Leu Ala Leu Asp Ser Pro Glu Ser Gly Trp 585590 595 aca gag gaa gat ggt ccc aaa gaa gga ctt gct gaa tac att gtt gag1880 Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala Glu Tyr Ile Val Glu 600605 610 ttt ctg aag aag aag gct gag atg ctt gca gac tat ttc tct ttg gaa1928 Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp Tyr Phe Ser Leu Glu 615620 625 att gat gag gaa ggg aac ctg att gga tta ccc ctt ctg att gac aac1976 Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro Leu Leu Ile Asp Asn 630635 640 645 tat gtg ccc cct ttg gag gga ctg cct atc ttc att ctt cga ctagcc 2024 Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe Ile Leu Arg Leu Ala650 655 660 act gag gtg aat tgg gac gaa gaa aag gaa tgt ttt gaa agc ctcagt 2072 Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys Phe Glu Ser Leu Ser665 670 675 aaa gaa tgc gct atg ttc tat tcc atc cgg aag cag tac ata tctgag 2120 Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys Gln Tyr Ile Ser Glu680 685 690 gag tcg acc ctc tca ggc cag cag agt gaa gtg cct ggc tcc attcca 2168 Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val Pro Gly Ser Ile Pro695 700 705 aac tcc tgg aag tgg act gtg gaa cac att gtc tat aaa gcc ttgcgc 2216 Asn Ser Trp Lys Trp Thr Val Glu His Ile Val Tyr Lys Ala Leu Arg710 715 720 725 tca cac att ctg cct cct aaa cat ttc aca gaa gat gga aatatc ctg 2264 Ser His Ile Leu Pro Pro Lys His Phe Thr Glu Asp Gly Asn IleLeu 730 735 740 cag ctt gct aac ctg cct gat cta tac aaa gtc ttt gag aggtgt taa 2312 Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg Cys745 750 755 atatggttat ttatgcactg tgggatgtgt tcttctttct ctgtattccgatacaaagtg 2372 ttgtatcaaa gtgtgatata caaagtgtac caacataagt gttggtagcacttaagactt 2432 atacttgcct tctgatagta ttcctttata cacagtggat tgattataaataaatagatg 2492 tgtcttaaca taaaaaaaaa aaaaaaaaaa aaa 2525 2 756 PRT homosapiens 2 Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr ValVal 1 5 10 15 Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala AsnAla Ile 20 25 30 Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr SerIle Gln 35 40 45 Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile GlnAsp Asn 50 55 60 Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys GluArg Phe 65 70 75 80 Thr Thr Ser Lys Leu Gln Ser Phe Glu Asp Leu Ala SerIle Ser Thr 85 90 95 Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser HisVal Ala His 100 105 110 Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys CysAla Tyr Arg Ala 115 120 125 Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro ProLys Pro Cys Ala Gly 130 135 140 Asn Gln Gly Thr Gln Ile Thr Val Glu AspLeu Phe Tyr Asn Ile Ala 145 150 155 160 Thr Arg Arg Lys Ala Leu Lys AsnPro Ser Glu Glu Tyr Gly Lys Ile 165 170 175 Leu Glu Val Val Gly Arg TyrSer Val His Asn Ala Gly Ile Ser Phe 180 185 190 Ser Val Lys Lys Gln GlyGlu Thr Val Ala Asp Val Arg Thr Leu Pro 195 200 205 Asn Ala Ser Thr ValAsp Asn Ile Arg Ser Val Phe Gly Asn Ala Val 210 215 220 Ser Arg Glu LeuIle Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 225 230 235 240 Lys MetAsn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys 245 250 255 IlePhe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu 260 265 270Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys Asn Thr 275 280285 His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln Asn Val Asp 290295 300 Val Asn Val His Pro Thr Lys His Glu Val His Phe Leu His Glu Glu305 310 315 320 Ser Ile Leu Glu Arg Val Gln Gln His Ile Glu Ser Lys LeuLeu Gly 325 330 335 Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Thr Leu LeuPro Gly Leu 340 345 350 Ala Gly Pro Ser Gly Glu Met Val Lys Ser Thr ThrSer Leu Thr Ser 355 360 365 Ser Ser Thr Ser Gly Ser Ser Asp Lys Val TyrAla His Gln Met Val 370 375 380 Arg Thr Asp Ser Arg Glu Gln Lys Leu AspAla Phe Leu Gln Pro Leu 385 390 395 400 Ser Lys Pro Leu Ser Ser Gln ProGln Ala Ile Val Thr Glu Asp Lys 405 410 415 Thr Asp Ile Ser Ser Gly ArgAla Arg Gln Gln Asp Glu Glu Met Leu 420 425 430 Glu Leu Pro Ala Pro AlaGlu Val Ala Ala Lys Asn Gln Ser Leu Glu 435 440 445 Gly Asp Thr Thr LysGly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro 450 455 460 Thr Ser Ser AsnPro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu 465 470 475 480 Met ValGlu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 485 490 495 ArgArg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu 500 505 510Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu His Asn His 515 520525 Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala Leu Ala Gln His Gln 530535 540 Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe545 550 555 560 Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn Phe Gly Val LeuArg Leu 565 570 575 Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Met Leu AlaLeu Asp Ser 580 585 590 Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pro LysGlu Gly Leu Ala 595 600 605 Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys AlaGlu Met Leu Ala Asp 610 615 620 Tyr Phe Ser Leu Glu Ile Asp Glu Glu GlyAsn Leu Ile Gly Leu Pro 625 630 635 640 Leu Leu Ile Asp Asn Tyr Val ProPro Leu Glu Gly Leu Pro Ile Phe 645 650 655 Ile Leu Arg Leu Ala Thr GluVal Asn Trp Asp Glu Glu Lys Glu Cys 660 665 670 Phe Glu Ser Leu Ser LysGlu Cys Ala Met Phe Tyr Ser Ile Arg Lys 675 680 685 Gln Tyr Ile Ser GluGlu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 690 695 700 Pro Gly Ser IlePro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val 705 710 715 720 Tyr LysAla Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 725 730 735 AspGly Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val 740 745 750Phe Glu Arg Cys 755 3 3063 DNA homo sapiens CDS (81)..(2879) 3ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60ctgctctgtt aaaagcgaaa atg aaa caa ttg cct gcg gca aca gtt cga ctc 113Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu 1 5 10 ctt tca agt tct cagatc atc act tcg gtg gtc agt gtt gta aaa gag 161 Leu Ser Ser Ser Gln IleIle Thr Ser Val Val Ser Val Val Lys Glu 15 20 25 ctt att gaa aac tcc ttggat gct ggt gcc aca agc gta gat gtt aaa 209 Leu Ile Glu Asn Ser Leu AspAla Gly Ala Thr Ser Val Asp Val Lys 30 35 40 ctg gag aac tat gga ttt gataaa att gag gtg cga gat aac ggg gag 257 Leu Glu Asn Tyr Gly Phe Asp LysIle Glu Val Arg Asp Asn Gly Glu 45 50 55 ggt atc aag gct gtt gat gca cctgta atg gca atg aag tac tac acc 305 Gly Ile Lys Ala Val Asp Ala Pro ValMet Ala Met Lys Tyr Tyr Thr 60 65 70 75 tca aaa ata aat agt cat gaa gatctt gaa aat ttg aca act tac ggt 353 Ser Lys Ile Asn Ser His Glu Asp LeuGlu Asn Leu Thr Thr Tyr Gly 80 85 90 ttt cgt gga gaa gcc ttg ggg tca atttgt tgt ata gct gag gtt tta 401 Phe Arg Gly Glu Ala Leu Gly Ser Ile CysCys Ile Ala Glu Val Leu 95 100 105 att aca aca aga acg gct gct gat aatttt agc acc cag tat gtt tta 449 Ile Thr Thr Arg Thr Ala Ala Asp Asn PheSer Thr Gln Tyr Val Leu 110 115 120 gat ggc agt ggc cac ata ctt tct cagaaa cct tca cat ctt ggt caa 497 Asp Gly Ser Gly His Ile Leu Ser Gln LysPro Ser His Leu Gly Gln 125 130 135 ggt aca act gta act gct tta aga ttattt aag aat cta cct gta aga 545 Gly Thr Thr Val Thr Ala Leu Arg Leu PheLys Asn Leu Pro Val Arg 140 145 150 155 aag cag ttt tac tca act gca aaaaaa tgt aaa gat gaa ata aaa aag 593 Lys Gln Phe Tyr Ser Thr Ala Lys LysCys Lys Asp Glu Ile Lys Lys 160 165 170 atc caa gat ctc ctc atg agc tttggt atc ctt aaa cct gac tta agg 641 Ile Gln Asp Leu Leu Met Ser Phe GlyIle Leu Lys Pro Asp Leu Arg 175 180 185 att gtc ttt gta cat aac aag gcagtt att tgg cag aaa agc aga gta 689 Ile Val Phe Val His Asn Lys Ala ValIle Trp Gln Lys Ser Arg Val 190 195 200 tca gat cac aag atg gct ctc atgtca gtt ctg ggg act gct gtt atg 737 Ser Asp His Lys Met Ala Leu Met SerVal Leu Gly Thr Ala Val Met 205 210 215 aac aat atg gaa tcc ttt cag taccac tct gaa gaa tct cag att tat 785 Asn Asn Met Glu Ser Phe Gln Tyr HisSer Glu Glu Ser Gln Ile Tyr 220 225 230 235 ctc agt gga ttt ctt cca aagtgt gat gca gac cac tct ttc act agt 833 Leu Ser Gly Phe Leu Pro Lys CysAsp Ala Asp His Ser Phe Thr Ser 240 245 250 ctt tca aca cca gaa aga agtttc atc ttc ata aac agt cga cca gta 881 Leu Ser Thr Pro Glu Arg Ser PheIle Phe Ile Asn Ser Arg Pro Val 255 260 265 cat caa aaa gat atc tta aagtta atc cga cat cat tac aat ctg aaa 929 His Gln Lys Asp Ile Leu Lys LeuIle Arg His His Tyr Asn Leu Lys 270 275 280 tgc cta aag gaa tct act cgtttg tat cct gtt ttc ttt ctg aaa atc 977 Cys Leu Lys Glu Ser Thr Arg LeuTyr Pro Val Phe Phe Leu Lys Ile 285 290 295 gat gtt cct aca gct gat gttgat gta aat tta aca cca gat aaa agc 1025 Asp Val Pro Thr Ala Asp Val AspVal Asn Leu Thr Pro Asp Lys Ser 300 305 310 315 caa gta tta tta caa aataag gaa tct gtt tta att gct ctt gaa aat 1073 Gln Val Leu Leu Gln Asn LysGlu Ser Val Leu Ile Ala Leu Glu Asn 320 325 330 ctg atg acg act tgt tatgga cca tta cct agt aca aat tct tat gaa 1121 Leu Met Thr Thr Cys Tyr GlyPro Leu Pro Ser Thr Asn Ser Tyr Glu 335 340 345 aat aat aaa aca gat gtttcc gca gct gac atc gtt ctt agt aaa aca 1169 Asn Asn Lys Thr Asp Val SerAla Ala Asp Ile Val Leu Ser Lys Thr 350 355 360 gca gaa aca gat gtg cttttt aat aaa gtg gaa tca tct gga aag aat 1217 Ala Glu Thr Asp Val Leu PheAsn Lys Val Glu Ser Ser Gly Lys Asn 365 370 375 tat tca aat gtt gat acttca gtc att cca ttc caa aat gat atg cat 1265 Tyr Ser Asn Val Asp Thr SerVal Ile Pro Phe Gln Asn Asp Met His 380 385 390 395 aat gat gaa tct ggaaaa aac act gat gat tgt tta aat cac cag ata 1313 Asn Asp Glu Ser Gly LysAsn Thr Asp Asp Cys Leu Asn His Gln Ile 400 405 410 agt att ggt gac tttggt tat ggt cat tgt agt agt gaa att tct aac 1361 Ser Ile Gly Asp Phe GlyTyr Gly His Cys Ser Ser Glu Ile Ser Asn 415 420 425 att gat aaa aac actaag aat gca ttt cag gac att tca atg agt aat 1409 Ile Asp Lys Asn Thr LysAsn Ala Phe Gln Asp Ile Ser Met Ser Asn 430 435 440 gta tca tgg gag aactct cag acg gaa tat agt aaa act tgt ttt ata 1457 Val Ser Trp Glu Asn SerGln Thr Glu Tyr Ser Lys Thr Cys Phe Ile 445 450 455 agt tcc gtt aag cacacc cag tca gaa aat ggc aat aaa gac cat ata 1505 Ser Ser Val Lys His ThrGln Ser Glu Asn Gly Asn Lys Asp His Ile 460 465 470 475 gat gag agt ggggaa aat gag gaa gaa gca ggt ctt gaa aac tct tcg 1553 Asp Glu Ser Gly GluAsn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser 480 485 490 gaa att tct gcagat gag tgg agc agg gga aat ata ctt aaa aat tca 1601 Glu Ile Ser Ala AspGlu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser 495 500 505 gtg gga gag aatatt gaa cct gtg aaa att tta gtg cct gaa aaa agt 1649 Val Gly Glu Asn IleGlu Pro Val Lys Ile Leu Val Pro Glu Lys Ser 510 515 520 tta cca tgt aaagta agt aat aat aat tat cca atc cct gaa caa atg 1697 Leu Pro Cys Lys ValSer Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met 525 530 535 aat ctt aat gaagat tca tgt aac aaa aaa tca aat gta ata gat aat 1745 Asn Leu Asn Glu AspSer Cys Asn Lys Lys Ser Asn Val Ile Asp Asn 540 545 550 555 aaa tct ggaaaa gtt aca gct tat gat tta ctt agc aat cga gta atc 1793 Lys Ser Gly LysVal Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile 560 565 570 aag aaa cccatg tca gca agt gct ctt ttt gtt caa gat cat cgt cct 1841 Lys Lys Pro MetSer Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro 575 580 585 cag ttt ctcata gaa aat cct aag act agt tta gag gat gca aca cta 1889 Gln Phe Leu IleGlu Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu 590 595 600 caa att gaagaa ctg tgg aag aca ttg agt gaa gag gaa aaa ctg aaa 1937 Gln Ile Glu GluLeu Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys 605 610 615 tat gaa gagaag gct act aaa gac ttg gaa cga tac aat agt caa atg 1985 Tyr Glu Glu LysAla Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met 620 625 630 635 aag agagcc att gaa cag gag tca caa atg tca cta aaa gat ggc aga 2033 Lys Arg AlaIle Glu Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg 640 645 650 aaa aagata aaa ccc acc agc gca tgg aat ttg gcc cag aag cac aag 2081 Lys Lys IleLys Pro Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys 655 660 665 tta aaaacc tca tta tct aat caa cca aaa ctt gat gaa ctc ctt cag 2129 Leu Lys ThrSer Leu Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln 670 675 680 tcc caaatt gaa aaa aga agg agt caa aat att aaa atg gta cag atc 2177 Ser Gln IleGlu Lys Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile 685 690 695 ccc ttttct atg aaa aac tta aaa ata aat ttt aag aaa caa aac aaa 2225 Pro Phe SerMet Lys Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys 700 705 710 715 gttgac tta gaa gag aag gat gaa cct tgc ttg atc cac aat ctc agg 2273 Val AspLeu Glu Glu Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg 720 725 730 tttcct gat gca tgg cta atg aca tcc aaa aca gag gta atg tta tta 2321 Phe ProAsp Ala Trp Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu 735 740 745 aatcca tat aga gta gaa gaa gcc ctg cta ttt aaa aga ctt ctt gag 2369 Asn ProTyr Arg Val Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu 750 755 760 aatcat aaa ctt cct gca gag cca ctg gaa aag cca att atg tta aca 2417 Asn HisLys Leu Pro Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr 765 770 775 gagagt ctt ttt aat gga tct cat tat tta gac gtt tta tat aaa atg 2465 Glu SerLeu Phe Asn Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met 780 785 790 795aca gca gat gac caa aga tac agt gga tca act tac ctg tct gat cct 2513 ThrAla Asp Asp Gln Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro 800 805 810cgt ctt aca gcg aat ggt ttc aag ata aaa ttg ata cca gga gtt tca 2561 ArgLeu Thr Ala Asn Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser 815 820 825att act gaa aat tac ttg gaa ata gaa gga atg gct aat tgt ctc cca 2609 IleThr Glu Asn Tyr Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro 830 835 840ttc tat gga gta gca gat tta aaa gaa att ctt aat gct ata tta aac 2657 PheTyr Gly Val Ala Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn 845 850 855aga aat gca aag gaa gtt tat gaa tgt aga cct cgc aaa gtg ata agt 2705 ArgAsn Ala Lys Glu Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser 860 865 870875 tat tta gag gga gaa gca gtg cgt cta tcc aga caa tta ccc atg tac 2753Tyr Leu Glu Gly Glu Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr 880 885890 tta tca aaa gag gac atc caa gac att atc tac aga atg aag cac cag 2801Leu Ser Lys Glu Asp Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln 895 900905 ttt gga aat gaa att aaa gag tgt gtt cat ggt cgc cca ttt ttt cat 2849Phe Gly Asn Glu Ile Lys Glu Cys Val His Gly Arg Pro Phe Phe His 910 915920 cat tta acc tat ctt cca gaa act aca tga ttaaatatgt ttaagaagat 2899His Leu Thr Tyr Leu Pro Glu Thr Thr 925 930 tagttaccat tgaaattggttctgtcataa aacagcatga gtctggtttt aaattatctt 2959 tgtattatgt gtcacatggttattttttaa atgaggattc actgacttgt ttttatattg 3019 aaaaaagttc cacgtattgtagaaaacgta aataaactaa taac 3063 4 932 PRT homo sapiens 4 Met Lys Gln LeuPro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile ThrSer Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu Asp AlaGly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40 45 Phe Asp LysIle Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 50 55 60 Asp Ala ProVal Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 65 70 75 80 His GluAsp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 85 90 95 Leu GlySer Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 100 105 110 AlaAla Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 115 120 125Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 130 135140 Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145150 155 160 Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp LeuLeu 165 170 175 Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val PheVal His 180 185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser AspHis Lys Met 195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val Met AsnAsn Met Glu Ser 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile TyrLeu Ser Gly Phe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His Ser PheThr Ser Leu Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe Ile Asn SerArg Pro Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu Ile Arg His HisTyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr Arg Leu Tyr Pro ValPhe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295 300 Asp Val Asp Val AsnLeu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 305 310 315 320 Asn Lys GluSer Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 325 330 335 Tyr GlyPro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 340 345 350 ValSer Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 355 360 365Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 370 375380 Thr Ser Val Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 385390 395 400 Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly AspPhe 405 410 415 Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp LysAsn Thr 420 425 430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val SerTrp Glu Asn 435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile SerSer Val Lys His 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His IleAsp Glu Ser Gly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu AsnSer Ser Glu Ile Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn Ile LeuLys Asn Ser Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys Ile Leu ValPro Glu Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn Asn Asn Tyr ProIle Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540 Ser Cys Asn Lys LysSer Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545 550 555 560 Thr Ala TyrAsp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 565 570 575 Ala SerAla Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 580 585 590 AsnPro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 595 600 605Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 610 615620 Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 625630 635 640 Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile LysPro 645 650 655 Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys ThrSer Leu 660 665 670 Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser GlnIle Glu Lys 675 680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile ProPhe Ser Met Lys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn LysVal Asp Leu Glu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile His AsnLeu Arg Phe Pro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu ValMet Leu Leu Asn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu Phe LysArg Leu Leu Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro Leu Glu LysPro Ile Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly Ser His Tyr LeuAsp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790 795 800 Arg Tyr SerGly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 805 810 815 Gly PheLys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 820 825 830 LeuGlu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 835 840 845Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 850 855860 Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 865870 875 880 Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys GluAsp 885 890 895 Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly AsnGlu Ile 900 905 910 Lys Glu Cys Val His Gly Arg Pro Phe Phe His His LeuThr Tyr Leu 915 920 925 Pro Glu Thr Thr 930 5 2771 DNA homo sapiens CDS(25)..(2613) 5 cgaggcggat cgggtgttgc atcc atg gag cga gct gag agc tcgagt aca 51 Met Glu Arg Ala Glu Ser Ser Ser Thr 1 5 gaa cct gct aag gccatc aaa cct att gat cgg aag tca gtc cat cag 99 Glu Pro Ala Lys Ala IleLys Pro Ile Asp Arg Lys Ser Val His Gln 10 15 20 25 att tgc tct ggg caggtg gta ctg agt cta agc act gcg gta aag gag 147 Ile Cys Ser Gly Gln ValVal Leu Ser Leu Ser Thr Ala Val Lys Glu 30 35 40 tta gta gaa aac agt ctggat gct ggt gcc act aat att gat cta aag 195 Leu Val Glu Asn Ser Leu AspAla Gly Ala Thr Asn Ile Asp Leu Lys 45 50 55 ctt aag gac tat gga gtg gatctt att gaa gtt tca gac aat gga tgt 243 Leu Lys Asp Tyr Gly Val Asp LeuIle Glu Val Ser Asp Asn Gly Cys 60 65 70 ggg gta gaa gaa gaa aac ttc gaaggc tta act ctg aaa cat cac aca 291 Gly Val Glu Glu Glu Asn Phe Glu GlyLeu Thr Leu Lys His His Thr 75 80 85 tct aag att caa gag ttt gcc gac ctaact cag gtt gaa act ttt ggc 339 Ser Lys Ile Gln Glu Phe Ala Asp Leu ThrGln Val Glu Thr Phe Gly 90 95 100 105 ttt cgg ggg gaa gct ctg agc tcactt tgt gca ctg agc gat gtc acc 387 Phe Arg Gly Glu Ala Leu Ser Ser LeuCys Ala Leu Ser Asp Val Thr 110 115 120 att tct acc tgc cac gca tcg gcgaag gtt gga act cga ctg atg ttt 435 Ile Ser Thr Cys His Ala Ser Ala LysVal Gly Thr Arg Leu Met Phe 125 130 135 gat cac aat ggg aaa att atc cagaaa acc ccc tac ccc cgc ccc aga 483 Asp His Asn Gly Lys Ile Ile Gln LysThr Pro Tyr Pro Arg Pro Arg 140 145 150 ggg acc aca gtc agc gtg cag cagtta ttt tcc aca cta cct gtg cgc 531 Gly Thr Thr Val Ser Val Gln Gln LeuPhe Ser Thr Leu Pro Val Arg 155 160 165 cat aag gaa ttt caa agg aat attaag aag gag tat gcc aaa atg gtc 579 His Lys Glu Phe Gln Arg Asn Ile LysLys Glu Tyr Ala Lys Met Val 170 175 180 185 cag gtc tta cat gca tac tgtatc att tca gca ggc atc cgt gta agt 627 Gln Val Leu His Ala Tyr Cys IleIle Ser Ala Gly Ile Arg Val Ser 190 195 200 tgc acc aat cag ctt gga caagga aaa cga cag cct gtg gta tgc aca 675 Cys Thr Asn Gln Leu Gly Gln GlyLys Arg Gln Pro Val Val Cys Thr 205 210 215 ggt gga agc ccc agc ata aaggaa aat atc ggc tct gtg ttt ggg cag 723 Gly Gly Ser Pro Ser Ile Lys GluAsn Ile Gly Ser Val Phe Gly Gln 220 225 230 aag cag ttg caa agc ctc attcct ttt gtt cag ctg ccc cct agt gac 771 Lys Gln Leu Gln Ser Leu Ile ProPhe Val Gln Leu Pro Pro Ser Asp 235 240 245 tcc gtg tgt gaa gag tac ggtttg agc tgt tcg gat gct ctg cat aat 819 Ser Val Cys Glu Glu Tyr Gly LeuSer Cys Ser Asp Ala Leu His Asn 250 255 260 265 ctt ttt tac atc tca ggtttc att tca caa tgc acg cat gga gtt gga 867 Leu Phe Tyr Ile Ser Gly PheIle Ser Gln Cys Thr His Gly Val Gly 270 275 280 agg agt tca aca gac agacag ttt ttc ttt atc aac cgg cgg cct tgt 915 Arg Ser Ser Thr Asp Arg GlnPhe Phe Phe Ile Asn Arg Arg Pro Cys 285 290 295 gac cca gca aag gtc tgcaga ctc gtg aat gag gtc tac cac atg tat 963 Asp Pro Ala Lys Val Cys ArgLeu Val Asn Glu Val Tyr His Met Tyr 300 305 310 aat cga cac cag tat ccattt gtt gtt ctt aac att tct gtt gat tca 1011 Asn Arg His Gln Tyr Pro PheVal Val Leu Asn Ile Ser Val Asp Ser 315 320 325 gaa tgc gtt gat atc aatgtt act cca gat aaa agg caa att ttg cta 1059 Glu Cys Val Asp Ile Asn ValThr Pro Asp Lys Arg Gln Ile Leu Leu 330 335 340 345 caa gag gaa aag cttttg ttg gca gtt tta aag acc tct ttg ata gga 1107 Gln Glu Glu Lys Leu LeuLeu Ala Val Leu Lys Thr Ser Leu Ile Gly 350 355 360 atg ttt gat agt gatgtc aac aag cta aat gtc agt cag cag cca ctg 1155 Met Phe Asp Ser Asp ValAsn Lys Leu Asn Val Ser Gln Gln Pro Leu 365 370 375 ctg gat gtt gaa ggtaac tta ata aaa atg cat gca gcg gat ttg gaa 1203 Leu Asp Val Glu Gly AsnLeu Ile Lys Met His Ala Ala Asp Leu Glu 380 385 390 aag ccc atg gta gaaaag cag gat caa tcc cct tca tta agg act gga 1251 Lys Pro Met Val Glu LysGln Asp Gln Ser Pro Ser Leu Arg Thr Gly 395 400 405 gaa gaa aaa aaa gacgtg tcc att tcc aga ctg cga gag gcc ttt tct 1299 Glu Glu Lys Lys Asp ValSer Ile Ser Arg Leu Arg Glu Ala Phe Ser 410 415 420 425 ctt cgt cac acaaca gag aac aag cct cac agc cca aag act cca gaa 1347 Leu Arg His Thr ThrGlu Asn Lys Pro His Ser Pro Lys Thr Pro Glu 430 435 440 cca aga agg agccct cta gga cag aaa agg ggt atg ctg tct tct agc 1395 Pro Arg Arg Ser ProLeu Gly Gln Lys Arg Gly Met Leu Ser Ser Ser 445 450 455 act tca ggt gccatc tct gac aaa ggc gtc ctg aga cct cag aaa gag 1443 Thr Ser Gly Ala IleSer Asp Lys Gly Val Leu Arg Pro Gln Lys Glu 460 465 470 gca gtg agt tccagt cac gga ccc agt gac cct acg gac aga gcg gag 1491 Ala Val Ser Ser SerHis Gly Pro Ser Asp Pro Thr Asp Arg Ala Glu 475 480 485 gtg gag aag gactcg ggg cac ggc agc act tcc gtg gat tct gag ggg 1539 Val Glu Lys Asp SerGly His Gly Ser Thr Ser Val Asp Ser Glu Gly 490 495 500 505 ttc agc atccca gac acg ggc agt cac tgc agc agc gag tat gcg gcc 1587 Phe Ser Ile ProAsp Thr Gly Ser His Cys Ser Ser Glu Tyr Ala Ala 510 515 520 agc tcc ccaggg gac agg ggc tcg cag gaa cat gtg gac tct cag gag 1635 Ser Ser Pro GlyAsp Arg Gly Ser Gln Glu His Val Asp Ser Gln Glu 525 530 535 aaa gcg cctgaa act gac gac tct ttt tca gat gtg gac tgc cat tca 1683 Lys Ala Pro GluThr Asp Asp Ser Phe Ser Asp Val Asp Cys His Ser 540 545 550 aac cag gaagat acc gga tgt aaa ttt cga gtt ttg cct cag cca act 1731 Asn Gln Glu AspThr Gly Cys Lys Phe Arg Val Leu Pro Gln Pro Thr 555 560 565 aat ctc gcaacc cca aac aca aag cgt ttt aaa aaa gaa gaa att ctt 1779 Asn Leu Ala ThrPro Asn Thr Lys Arg Phe Lys Lys Glu Glu Ile Leu 570 575 580 585 tcc agttct gac att tgt caa aag tta gta aat act cag gac atg tca 1827 Ser Ser SerAsp Ile Cys Gln Lys Leu Val Asn Thr Gln Asp Met Ser 590 595 600 gcc tctcag gtt gat gta gct gtg aaa att aat aag aaa gtt gtg ccc 1875 Ala Ser GlnVal Asp Val Ala Val Lys Ile Asn Lys Lys Val Val Pro 605 610 615 ctg gacttt tct atg agt tct tta gct aaa cga ata aag cag tta cat 1923 Leu Asp PheSer Met Ser Ser Leu Ala Lys Arg Ile Lys Gln Leu His 620 625 630 cat gaagca cag caa agt gaa ggg gaa cag aat tac agg aag ttt agg 1971 His Glu AlaGln Gln Ser Glu Gly Glu Gln Asn Tyr Arg Lys Phe Arg 635 640 645 gca aagatt tgt cct gga gaa aat caa gca gcc gaa gat gaa cta aga 2019 Ala Lys IleCys Pro Gly Glu Asn Gln Ala Ala Glu Asp Glu Leu Arg 650 655 660 665 aaagag ata agt aaa acg atg ttt gca gaa atg gaa atc att ggt cag 2067 Lys GluIle Ser Lys Thr Met Phe Ala Glu Met Glu Ile Ile Gly Gln 670 675 680 tttaac ctg gga ttt ata ata acc aaa ctg aat gag gat atc ttc ata 2115 Phe AsnLeu Gly Phe Ile Ile Thr Lys Leu Asn Glu Asp Ile Phe Ile 685 690 695 gtggac cag cat gcc acg gac gag aag tat aac ttc gag atg ctg cag 2163 Val AspGln His Ala Thr Asp Glu Lys Tyr Asn Phe Glu Met Leu Gln 700 705 710 cagcac acc gtg ctc cag ggg cag agg ctc ata gca cct cag act ctc 2211 Gln HisThr Val Leu Gln Gly Gln Arg Leu Ile Ala Pro Gln Thr Leu 715 720 725 aactta act gct gtt aat gaa gct gtt ctg ata gaa aat ctg gaa ata 2259 Asn LeuThr Ala Val Asn Glu Ala Val Leu Ile Glu Asn Leu Glu Ile 730 735 740 745ttt aga aag aat ggc ttt gat ttt gtt atc gat gaa aat gct cca gtc 2307 PheArg Lys Asn Gly Phe Asp Phe Val Ile Asp Glu Asn Ala Pro Val 750 755 760act gaa agg gct aaa ctg att tcc ttg cca act agt aaa aac tgg acc 2355 ThrGlu Arg Ala Lys Leu Ile Ser Leu Pro Thr Ser Lys Asn Trp Thr 765 770 775ttc gga ccc cag gac gtc gat gaa ctg atc ttc atg ctg agc gac agc 2403 PheGly Pro Gln Asp Val Asp Glu Leu Ile Phe Met Leu Ser Asp Ser 780 785 790cct ggg gtc atg tgc cgg cct tcc cga gtc aag cag atg ttt gcc tcc 2451 ProGly Val Met Cys Arg Pro Ser Arg Val Lys Gln Met Phe Ala Ser 795 800 805aga gcc tgc cgg aag tcg gtg atg att ggg act gct ctt aac aca agc 2499 ArgAla Cys Arg Lys Ser Val Met Ile Gly Thr Ala Leu Asn Thr Ser 810 815 820825 gag atg aag aaa ctg atc acc cac atg ggg gag atg gac cac ccc tgg 2547Glu Met Lys Lys Leu Ile Thr His Met Gly Glu Met Asp His Pro Trp 830 835840 aac tgt ccc cat gga agg cca acc atg aga cac atc gcc aac ctg ggt 2595Asn Cys Pro His Gly Arg Pro Thr Met Arg His Ile Ala Asn Leu Gly 845 850855 gtc att tct cag aac tga ccgtagtcac tgtatggaat aattggtttt 2643 ValIle Ser Gln Asn 860 atcgcagatt tttatgtttt gaaagacaga gtcttcactaaccttttttg ttttaaaatg 2703 aaacctgcta cttaaaaaaa atacacatca cacccatttaaaagtgatct tgagaacctt 2763 ttcaaacc 2771 6 862 PRT homo sapiens 6 MetGlu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 1 5 10 15Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 20 25 30Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 7580 Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85 9095 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 100105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser115 120 125 Ala Lys Val Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys IleIle 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val SerVal Gln 145 150 155 160 Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys GluPhe Gln Arg Asn 165 170 175 Ile Lys Lys Glu Tyr Ala Lys Met Val Gln ValLeu His Ala Tyr Cys 180 185 190 Ile Ile Ser Ala Gly Ile Arg Val Ser CysThr Asn Gln Leu Gly Gln 195 200 205 Gly Lys Arg Gln Pro Val Val Cys ThrGly Gly Ser Pro Ser Ile Lys 210 215 220 Glu Asn Ile Gly Ser Val Phe GlyGln Lys Gln Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe Val Gln Leu ProPro Ser Asp Ser Val Cys Glu Glu Tyr Gly 245 250 255 Leu Ser Cys Ser AspAla Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 260 265 270 Ile Ser Gln CysThr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln 275 280 285 Phe Phe PheIle Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg 290 295 300 Leu ValAsn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320Val Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 340345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Val Asn355 360 365 Lys Leu Asn Val Ser Gln Gln Pro Leu Leu Asp Val Glu Gly AsnLeu 370 375 380 Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val GluLys Gln 385 390 395 400 Asp Gln Ser Pro Ser Leu Arg Thr Gly Glu Glu LysLys Asp Val Ser 405 410 415 Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu ArgHis Thr Thr Glu Asn 420 425 430 Lys Pro His Ser Pro Lys Thr Pro Glu ProArg Arg Ser Pro Leu Gly 435 440 445 Gln Lys Arg Gly Met Leu Ser Ser SerThr Ser Gly Ala Ile Ser Asp 450 455 460 Lys Gly Val Leu Arg Pro Gln LysGlu Ala Val Ser Ser Ser His Gly 465 470 475 480 Pro Ser Asp Pro Thr AspArg Ala Glu Val Glu Lys Asp Ser Gly His 485 490 495 Gly Ser Thr Ser ValAsp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 500 505 510 Ser His Cys SerSer Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly 515 520 525 Ser Gln GluHis Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp 530 535 540 Ser PheSer Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys 545 550 555 560Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu Ala Thr Pro Asn Thr 565 570575 Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln 580585 590 Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala595 600 605 Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met SerSer 610 615 620 Leu Ala Lys Arg Ile Lys Gln Leu His His Glu Ala Gln GlnSer Glu 625 630 635 640 Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys IleCys Pro Gly Glu 645 650 655 Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys GluIle Ser Lys Thr Met 660 665 670 Phe Ala Glu Met Glu Ile Ile Gly Gln PheAsn Leu Gly Phe Ile Ile 675 680 685 Thr Lys Leu Asn Glu Asp Ile Phe IleVal Asp Gln His Ala Thr Asp 690 695 700 Glu Lys Tyr Asn Phe Glu Met LeuGln Gln His Thr Val Leu Gln Gly 705 710 715 720 Gln Arg Leu Ile Ala ProGln Thr Leu Asn Leu Thr Ala Val Asn Glu 725 730 735 Ala Val Leu Ile GluAsn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 740 745 750 Phe Val Ile AspGlu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 755 760 765 Ser Leu ProThr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val Asp 770 775 780 Glu LeuIle Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro 785 790 795 800Ser Arg Val Lys Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val 805 810815 Met Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr 820825 830 His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro835 840 845 Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gln Asn 850855 860 7 20 DNA Artificial Sequence hMLH1 sense primer 7 gttgaacatctagacgtctc 20 8 19 DNA Artificial Sequence hMLH1 sense primer 8tcgtggcagg ggttattcg 19 9 19 DNA Artificial Sequence hMLH1 sense primer9 ctacccaatg cctcaaccg 19 10 22 DNA Artificial Sequence hMLH1 senseprimer 10 gagaactgat agaaattgga tg 22 11 18 DNA Artificial SequencehMLH1 sense primer 11 gggacatgag gttctccg 18 12 19 DNA ArtificialSequence hMLH1 sense primer 12 gggctgtgtg aatcctcag 19 13 20 DNAArtificial Sequence hMLH1 antisense primer 13 cggttcacca ctgtctcgtc 2014 18 DNA Artificial Sequence hMLH1 antisense primer 14 tccaggatgctctcctcg 18 15 20 DNA Artificial Sequence hMLH1 antisense primer 15caagtcctgg tagcaaagtc 20 16 19 DNA Artificial Sequence hMLH1 antisenseprimer 16 atggcaaggt caaagagcg 19 17 22 DNA Artificial Sequence hMLH1antisense primer 17 caacaatgta ttcagnaagt cc 22 18 21 DNA ArtificialSequence hMLH1 antisense primer 18 ttgatacaac actttgtatc g 21 19 21 DNAArtificial Sequence hMLH1 antisense primer 19 ggaatactat cagaaggcaa g 2120 21 DNA Artificial Sequence hMLH1 sense primer 20 acagagcaagttactcagat g 21 21 20 DNA Artificial Sequence hMLH1 sense primer 21gtacacaatg caggcattag 20 22 21 DNA Artificial Sequence hMLH1 senseprimer 22 aatgtggatg ttaatgtgca c 21 23 18 DNA Artificial Sequence hMLH1sense primer 23 ctgacctcgt cttcctac 18 24 19 DNA Artificial SequencehMLH1 sense primer 24 cagcaagatg aggagatgc 19 25 21 DNA ArtificialSequence hMLH1 sense primer 25 ggaaatggtg gaagatgatt c 21 26 16 DNAArtificial Sequence hMLH1 sense primer 26 cttctcaaca ccaagc 16 27 21 DNAArtificial Sequence hMLH1 sense primer 27 gaaattgatg aggaagggaa c 21 2822 DNA Artificial Sequence hMLH1 sense primer 28 cttctgattg acaactatgtgc 22 29 22 DNA Artificial Sequence hMLH1 sense primer 29 cacagaagatggaaatatcc tg 22 30 20 DNA Artificial Sequence hMLH1 sense primer 30gtgttggtag cacttaagac 20 31 20 DNA Artificial Sequence hMLH1 antisenseprimer 31 tttcccatat tcttcacttg 20 32 19 DNA Artificial Sequence hMLH1antisense primer 32 gtaacatgag ccacatggc 19 33 19 DNA ArtificialSequence hMLH1 antisense primer 33 ccactgtctc gtccagccg 19 34 26 DNAArtificial Sequence hMLH1 5′ primer with BamHI restriction site 34cgggatccat gtcgttcgtg gcaggg 26 35 26 DNA Artificial Sequence hMLH1 3′primer with XbaI restriction site 35 gctctagatt aacacctctc aaagac 26 3621 DNA Artificial Sequence hMLH1 primer useful for amplifying codons 1to 394 36 gcatctagac gtttccttgg c 21 37 20 DNA Artificial Sequenceprimer useful for amplifying codons 1 to 394 of hMLH1 37 catccaagcttctgttcccg 20 38 19 DNA Artificial Sequence primer useful for amplifyingcodons 326 to 729 of hMLH1 38 ggggtgcagc agcacatcg 19 39 20 DNAArtificial Sequence primer useful for amplifying codons 326 to 729 ofhMLH1 39 ggaggcagaa tgtgtgagcg 20 40 19 DNA Artificial Sequence primeruseful for amplifying codons 602 to 756 plus 128 nucleotides of 3′untranslated sequence of hMLH1 40 tcccaaagaa ggacttgct 19 41 22 DNAArtificial Sequence primer useful for amplifying codons 602 to 756 plus128 nucleotides of 3′ untranslated sequence of hMLH1 41 agtataagtcttaagtgcta cc 22 42 20 DNA Artificial Sequence primer useful foramplifying codons 578 to 632 of hMLH1 42 tttatggttt ctcacctgcc 20 43 19DNA Artificial Sequence primer useful for amplifying codons 578 to 632of hMLH1 43 gttatctgcc cacctcagc 19 44 59 DNA Artificial Sequence primeruseful for amplifying codons 1 to 394 of hMLH1 wherein PCR product maybe used for coupled transcription- translation 44 ggatcctaat acgactcactatagggagac caccatggca tctagacgtt tcccttggc 59 45 20 DNA ArtificialSequence primer useful for amplifying codons 1 to 394 of hMLH1 whereinPCR product may be used for coupled transcription- translation 45catccaagct tctgttcccg 20 46 56 DNA Artificial Sequence primer useful foramplifying codons 326 to 729 of hMLH1 wherein PCR product may be usedfor coupled transcription- translation 46 ggatcctaat acgactcactatagggagac caccatgggg gtgcagcagc acatcg 56 47 20 DNA Artificial Sequenceprimer useful for amplifying codons 326 to 729 of hMLH1 wherein PCRproduct may be used for coupled transcription-translation 47 ggaggcagaatgtgtgagcg 20 48 28 DNA Artificial Sequence hMLH2 5′ primer with a BamHIrestriction site 48 cgggatccat gaaacaattg cctgcggc 28 49 26 DNAArtificial Sequence hMLH2 3′ primer with XbaI restriction site 49gctctagacc agactcatgc tgtttt 26 50 26 DNA Artificial Sequence hMLH3 5′primer with a BamHI restriction site 50 cgggatccat ggagcgagct gagagc 2651 23 DNA Artificial Sequence hMLH3 3′ primer with XbaI restriction site51 gctctagagt gaagactctg tct 23 52 20 DNA Artificial Sequence hMLH2primer 52 aagctgctct gttaaaagcg 20 53 18 DNA Artificial Sequence hMLH2primer 53 gcaccagcat ccaaggag 18 54 19 DNA Artificial Sequence hMLH3primer 54 caaccatgag acacatcgc 19 55 20 DNA Artificial Sequence hMLH3primer 55 aggttagtga agactctgtc 20 56 53 DNA Artificial Sequence primeruseful for amplifying codons 1 to 500 of hMLH2 56 ggatcctaat acgactcactatagggagac caccatggaa caattgcctg cgg 53 57 18 DNA Artificial Sequenceprimer useful for amplifying codons 1 to 500 of hMLH2 57 cctgctccactcatctgc 18 58 60 DNA Artificial Sequence primer useful for amplifyingcodons 270 to 755 of hMLH2 58 ggatcctaat acgactcact atagggagaccaccatggaa gatatcttaa agttaatccg 60 59 21 DNA Artificial Sequence primeruseful for amplifying codons 270 to 755 of hMLH2 59 ggcttcttctactctatatg g 21 60 58 DNA Artificial Sequence primer useful foramplifying from codon 485 to the translation termination site at codon933 of hMLH2 60 ggatcctaat acgactcact atagggagac caccatggca ggtcttgaaaactcttcg 58 61 21 DNA Artificial Sequence primer useful for amplifyingfrom codon 485 to the translation termination site at codon 933 of hMLH261 aaaacaagtc agtgaatcct c 21 62 20 DNA Artificial Sequence 3′ primeruseful for amplifying up to codon 369 of hMLH2 62 aagcacatct gtttctgctg20 63 20 DNA Artificial Sequence 3′ primer useful for amplifying up tocodon 290 of hMLH2 63 acgagtagat tcctttaggc 20 64 19 DNA ArtificialSequence 3′ primer useful for amplifying up to codon 214 of hMLH2 64cagaactgac atgagagcc 19 65 52 DNA Artificial Sequence primer useful foramplifying codons 1 to 863 hMLH3 65 ggatcctaat acgactcact atagggagaccaccatggag cgagctgaga gc 52 66 20 DNA Artificial Sequence primer usefulfor amplifying codons 1 to 863 hMLH3 66 aggttagtga agactctgtc 20 67 17DNA Artificial Sequence primer useful for amplifying up to codon 472 ofhMLH3 67 ctgaggtctc agcaggc 17 68 57 DNA Artificial Sequence primeruseful for amplifying codons 415 to 863 of hMLH3 68 ggatcctaatacgactcact atagggagac caccatggtg tccatttcca gactgcg 57 69 20 DNAArtificial Sequence primer useful for amplifying codons 415 to 863 ofhMLH3 69 aggttagtga agactctgtc 20 70 21 DNA Artificial Sequence primeruseful for amplifying codons 195 to 233 of hMLH2 70 ttatttggcagaaaagcaga g 21 71 21 DNA Artificial Sequence primer useful foramplifying codons 195 to 233 of hMLH2 71 ttaaaagact aacctcttgc c 21 7221 DNA Artificial Sequence sequencing primer useful for sequencingcodons 195 to 233 of hMLH2 72 ctgctgttat gaacaatatg g 21 73 19 DNAArtificial Sequence primer useful for amplifying codons 233 to 257 ofhMLH3 73 cagaagcagt tgcaaagcc 19 74 20 DNA Artificial Sequence primeruseful for amplifying codons 233 to 257 of hMLH3 74 aaaccgtactcttcacacac 20 75 20 DNA Artificial Sequence primer useful for amplifyingcodons 347 of 377 of hMLH3 75 gaggaaaagc ttttgttggc 20 76 18 DNAArtificial Sequence primer useful for amplifying codons 347 of 377 ofhMLH3 76 cagtggctgc tgactgac 18 77 19 DNA Artificial Sequence primeruseful for amplifying codons 439 to 472 of hMLH3 77 tccagaacca agaaggagc19 78 16 DNA Artificial Sequence primer useful for amplifying codons 439to 472 of hMLH3 78 tgaggtctca gcaggc 16

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding apolypeptide having the deduced amino acid sequence of SEQ ID NO:2 or afragment of said polypeptide; (b) a polynucleotide encoding apolypeptide having the amino acid sequence encoded by the cDNA containedin ATCC Deposit No. 75649; (c) a polynucleotide encoding a polypeptidehaving the deduced amino acid sequence of SEQ ID NO:4 or a fragment ofsaid polypeptide; (d) a polynucleotide encoding a polypeptide having theamino acid sequence encoded by the cDNA contained in ATCC Deposit No.75651; (e) a polynucleotide encoding a polypeptide having the deducedamino acid sequence of SEQ ID NO:6 or a fragment of said polypeptide;and (f) a polynucleotide encoding a polypeptide having the amino acidsequence encoded by the cDNA contained in ATCC Deposit No.
 75650. 2. Thepolynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. Thepolynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide having the deduced amino acid sequence of SEQ ID NO:2. 4.The polynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide having the deduced amino acid sequence of SEQ ID NO:4. 5.The polynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide having the deduced amino acid sequence of SEQ ID NO:6. 6.The polynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide encoded by the cDNA of ATCC Deposit No.
 75649. 7. Thepolynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide encoded by the cDNA of ATCC Deposit No.
 75651. 8. Thepolynucleotide of claim 1 wherein said polynucleotide encodes apolypeptide encoded by the cDNA of ATCC Deposit No.
 75650. 9. A vectorcontaining the polynucleotide of claim
 1. 10. A host cell geneticallyengineered with the vector of claim
 9. 11. A process for producing apolypeptide comprising expressing from the host cell of claim 10 thepolypeptide encoded by said DNA.
 12. A process for producing cellscapable of expressing a polypeptide comprising genetically engineeringcells with the vector of claim
 9. 13. A polypeptide comprising a memberselected from the group consisting of: (a) a polypeptide having thededuced amino acid sequence of SEQ ID NO:2 and fragments thereof; (b) apolypeptide encoded by the cDNA of ATCC Deposit No. 75649 and fragmentsof said polypeptide; (c) a polypeptide having the deduced amino acidsequence of SEQ ID NO:4 and fragments thereof; (d) a polypeptide encodedby the cDNA of ATCC Deposit No. 75651 and fragments of said polypeptide;(e) a polypeptide having the deduced amino acid sequence of SEQ ID NO:6and fragments thereof; and (f) a polypeptide encoded by the cDNA of ATCCDeposit No. 75650 and fragments of said polypeptide.
 14. An antibodythat specifically binds the polypeptide of claim 13.